PMID- 37929637 OWN - NLM STAT- Publisher LR - 20231108 IS - 1471-4159 (Electronic) IS - 0022-3042 (Linking) DP - 2023 Nov 6 TI - The dentate gyrus differentially metabolizes glucose and alternative fuels during rest and stimulation. LID - 10.1111/jnc.16004 [doi] AB - The metabolic demands of neuronal activity are both temporally and spatially dynamic, and neurons are particularly sensitive to disruptions in fuel and oxygen supply. Glucose is considered an obligate fuel for supporting brain metabolism. Although alternative fuels are often available, the extent of their contribution to central carbon metabolism remains debated. Differential fuel metabolism likely depends on cell type, location, and activity state, complicating its study. While biosensors provide excellent spatial and temporal information, they are limited to observations of only a few metabolites. On the other hand, mass spectrometry is rich in chemical information, but traditionally relies on cell culture or homogenized tissue samples. Here, we use mass spectrometry imaging (MALDI-MSI) to focus on the fuel metabolism of the dentate granule cell (DGC) layer in murine hippocampal slices. Using stable isotopes, we explore labeling dynamics at baseline, as well as in response to brief stimulation or fuel competition. We find that at rest, glucose is the predominant fuel metabolized through glycolysis, with little to no measurable contribution from glycerol or fructose. However, lactate/pyruvate, β-hydroxybutyrate (βHB), octanoate, and glutamine can contribute to TCA metabolism to varying degrees. In response to brief depolarization with 50 mM KCl, glucose metabolism was preferentially increased relative to the metabolism of alternative fuels. With an increased supply of alternative fuels, both lactate/pyruvate and βHB can outcompete glucose for TCA cycle entry. While lactate/pyruvate modestly reduced glucose contribution to glycolysis, βHB caused little change in glycolysis. This approach achieves broad metabolite coverage from a spatially defined region of physiological tissue, in which metabolic states are rapidly preserved following experimental manipulation. Using this powerful methodology, we investigated metabolism within the dentate gyrus not only at rest, but also in response to the energetic demand of activation, and in states of fuel competition. CI - © 2023 International Society for Neurochemistry. FAU - York, Elisa M AU - York EM AUID- ORCID: 0000-0001-8479-5231 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. FAU - Miller, Anne AU - Miller A AUID- ORCID: 0000-0002-5587-7497 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. FAU - Stopka, Sylwia A AU - Stopka SA AUID- ORCID: 0000-0003-3761-6899 AD - Department of Neurosurgery, Brigham and Women's Hospital, Boston, Massachusetts, USA. AD - Department of Radiology, Brigham and Women's Hospital, Boston, Massachusetts, USA. FAU - Martínez-François, Juan Ramón AU - Martínez-François JR AUID- ORCID: 0000-0002-1035-2574 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. FAU - Hossain, Md Amin AU - Hossain MA AUID- ORCID: 0000-0001-7136-503X AD - Department of Neurosurgery, Brigham and Women's Hospital, Boston, Massachusetts, USA. AD - Department of Radiology, Brigham and Women's Hospital, Boston, Massachusetts, USA. FAU - Baquer, Gerard AU - Baquer G AUID- ORCID: 0000-0002-4433-4972 AD - Department of Neurosurgery, Brigham and Women's Hospital, Boston, Massachusetts, USA. AD - Department of Radiology, Brigham and Women's Hospital, Boston, Massachusetts, USA. FAU - Regan, Michael S AU - Regan MS AUID- ORCID: 0000-0001-5252-2829 AD - Department of Neurosurgery, Brigham and Women's Hospital, Boston, Massachusetts, USA. AD - Department of Radiology, Brigham and Women's Hospital, Boston, Massachusetts, USA. FAU - Agar, Nathalie Y R AU - Agar NYR AUID- ORCID: 0000-0003-3149-3146 AD - Department of Neurosurgery, Brigham and Women's Hospital, Boston, Massachusetts, USA. AD - Department of Radiology, Brigham and Women's Hospital, Boston, Massachusetts, USA. AD - Department of Cancer Biology, Dana-Farber Cancer Institute; Harvard Medical School, Boston, Massachusetts, USA. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. LA - eng GR - R01 NS126248/NS/NINDS NIH HHS/United States GR - NS102586/NH/NIH HHS/United States GR - EY102586/NH/NIH HHS/United States GR - NS126248/NH/NIH HHS/United States PT - Journal Article DEP - 20231106 PL - England TA - J Neurochem JT - Journal of neurochemistry JID - 2985190R SB - IM OTO - NOTNLM OT - brain metabolism OT - glucose metabolism OT - ketone body metabolism OT - lactate metabolism OT - mass spectrometry imaging OT - stable isotope tracing EDAT- 2023/11/06 06:41 MHDA- 2023/11/06 06:41 CRDT- 2023/11/06 05:31 PHST- 2023/10/16 00:00 [revised] PHST- 2023/07/07 00:00 [received] PHST- 2023/10/21 00:00 [accepted] PHST- 2023/11/06 06:41 [medline] PHST- 2023/11/06 06:41 [pubmed] PHST- 2023/11/06 05:31 [entrez] AID - 10.1111/jnc.16004 [doi] PST - aheadofprint SO - J Neurochem. 2023 Nov 6. doi: 10.1111/jnc.16004. PMID- 37798473 OWN - NLM STAT- MEDLINE DCOM- 20231031 LR - 20240109 IS - 2522-5812 (Electronic) IS - 2522-5812 (Linking) VI - 5 IP - 10 DP - 2023 Oct TI - Spatially resolved metabolomics and isotope tracing reveal dynamic metabolic responses of dentate granule neurons with acute stimulation. PG - 1820-1835 LID - 10.1038/s42255-023-00890-z [doi] AB - Neuronal activity creates an intense energy demand that must be met by rapid metabolic responses. To investigate metabolic adaptations in the neuron-enriched dentate granule cell (DGC) layer within its native tissue environment, we employed murine acute hippocampal brain slices, coupled with fast metabolite preservation and followed by mass spectrometry (MS) imaging, to generate spatially resolved metabolomics and isotope-tracing data. Here we show that membrane depolarization induces broad metabolic changes, including increased glycolytic activity in DGCs. Increased glucose metabolism in response to stimulation is accompanied by mobilization of endogenous inosine into pentose phosphates via the action of purine nucleotide phosphorylase (PNP). The PNP reaction is an integral part of the neuronal response to stimulation, because inhibition of PNP leaves DGCs energetically impaired during recovery from strong activation. Performing MS imaging on brain slices bridges the gap between live-cell physiology and the deep chemical analysis enabled by MS. CI - © 2023. The Author(s), under exclusive licence to Springer Nature Limited. FAU - Miller, Anne AU - Miller A AUID- ORCID: 0000-0002-5587-7497 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. AD - Center for Pathobiochemistry and Genetics, Medical University of Vienna, Vienna, Austria. FAU - York, Elisa M AU - York EM AUID- ORCID: 0000-0001-8479-5231 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Stopka, Sylwia A AU - Stopka SA AUID- ORCID: 0000-0003-3761-6899 AD - Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. FAU - Martínez-François, Juan Ramón AU - Martínez-François JR AUID- ORCID: 0000-0002-1035-2574 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Hossain, Md Amin AU - Hossain MA AUID- ORCID: 0000-0001-7136-503X AD - Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. FAU - Baquer, Gerard AU - Baquer G AUID- ORCID: 0000-0002-4433-4972 AD - Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. FAU - Regan, Michael S AU - Regan MS AUID- ORCID: 0000-0001-5252-2829 AD - Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. FAU - Agar, Nathalie Y R AU - Agar NYR AUID- ORCID: 0000-0003-3149-3146 AD - Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. nathalie_agar@dfci.harvard.edu. AD - Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. nathalie_agar@dfci.harvard.edu. AD - Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. nathalie_agar@dfci.harvard.edu. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. gary_yellen@hms.harvard.edu. LA - eng GR - T32 EB025823/EB/NIBIB NIH HHS/United States GR - R37 NS102586/NS/NINDS NIH HHS/United States GR - R01 NS126248/NS/NINDS NIH HHS/United States GR - P30 EY012196/EY/NEI NIH HHS/United States GR - OT2 OD030544/OD/NIH HHS/United States GR - U2C DK119886/DK/NIDDK NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States GR - U54 CA210180/CA/NCI NIH HHS/United States PT - Journal Article DEP - 20231005 PL - Germany TA - Nat Metab JT - Nature metabolism JID - 101736592 RN - 0 (Isotopes) SB - IM UOF - Res Sq. 2023 Jul 25;:. PMID: 37546759 MH - Mice MH - Animals MH - *Dentate Gyrus/physiology MH - *Neurons MH - Cell Membrane MH - Isotopes MH - Metabolomics PMC - PMC10626993 MID - NIHMS1940573 COIS- DECLARATION OF INTERESTS N.Y.R.A. is a key opinion leader to Bruker. The other authors declare no competing interests. EDAT- 2023/10/06 00:43 MHDA- 2023/10/31 06:42 PMCR- 2024/04/05 CRDT- 2023/10/05 23:33 PHST- 2022/11/15 00:00 [received] PHST- 2023/08/09 00:00 [accepted] PHST- 2024/04/05 00:00 [pmc-release] PHST- 2023/10/31 06:42 [medline] PHST- 2023/10/06 00:43 [pubmed] PHST- 2023/10/05 23:33 [entrez] AID - 10.1038/s42255-023-00890-z [pii] AID - 10.1038/s42255-023-00890-z [doi] PST - ppublish SO - Nat Metab. 2023 Oct;5(10):1820-1835. doi: 10.1038/s42255-023-00890-z. Epub 2023 Oct 5. PMID- 36949206 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20230413 IS - 1476-4687 (Electronic) IS - 0028-0836 (Linking) VI - 616 IP - 7956 DP - 2023 Apr TI - Author Correction: Metabolic regulation of species-specific developmental rates. PG - E4 LID - 10.1038/s41586-023-05924-w [doi] FAU - Diaz-Cuadros, Margarete AU - Diaz-Cuadros M AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. mdiazcuadros@g.harvard.edu. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. mdiazcuadros@g.harvard.edu. AD - Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. mdiazcuadros@g.harvard.edu. FAU - Miettinen, Teemu P AU - Miettinen TP AUID- ORCID: 0000-0002-5975-200X AD - Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. FAU - Skinner, Owen S AU - Skinner OS AD - Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. AD - Department of Systems Biology, Harvard Medical School, Boston, MA, USA. AD - Broad Institute, Cambridge, MA, USA. FAU - Sheedy, Dylan AU - Sheedy D AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AUID- ORCID: 0000-0002-4352-2496 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. AD - Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. FAU - Gapon, Svetlana AU - Gapon S AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. FAU - Hubaud, Alexis AU - Hubaud A AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Manalis, Scott R AU - Manalis SR AUID- ORCID: 0000-0001-5223-9433 AD - Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. AD - Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. FAU - Oldham, William M AU - Oldham WM AUID- ORCID: 0000-0003-3029-4866 AD - Department of Medicine, Harvard Medical School, Boston, MA, USA. AD - Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. FAU - Pourquié, Olivier AU - Pourquié O AUID- ORCID: 0000-0001-5189-1227 AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. pourquie@genetics.med.harvard.edu. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. pourquie@genetics.med.harvard.edu. AD - Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA. pourquie@genetics.med.harvard.edu. LA - eng PT - Published Erratum PL - England TA - Nature JT - Nature JID - 0410462 SB - IM EFR - Nature. 2023 Jan;613(7944):550-557. PMID: 36599986 EDAT- 2023/03/24 06:00 MHDA- 2023/03/24 06:01 CRDT- 2023/03/23 00:21 PHST- 2023/03/24 06:01 [medline] PHST- 2023/03/24 06:00 [pubmed] PHST- 2023/03/23 00:21 [entrez] AID - 10.1038/s41586-023-05924-w [pii] AID - 10.1038/s41586-023-05924-w [doi] PST - ppublish SO - Nature. 2023 Apr;616(7956):E4. doi: 10.1038/s41586-023-05924-w. PMID- 36599986 OWN - NLM STAT- MEDLINE DCOM- 20230131 LR - 20230705 IS - 1476-4687 (Electronic) IS - 0028-0836 (Print) IS - 0028-0836 (Linking) VI - 613 IP - 7944 DP - 2023 Jan TI - Metabolic regulation of species-specific developmental rates. PG - 550-557 LID - 10.1038/s41586-022-05574-4 [doi] AB - Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development(1-3). However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD(+)/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD(+)/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies. CI - © 2023. The Author(s), under exclusive licence to Springer Nature Limited. FAU - Diaz-Cuadros, Margarete AU - Diaz-Cuadros M AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. mdiazcuadros@g.harvard.edu. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. mdiazcuadros@g.harvard.edu. AD - Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. mdiazcuadros@g.harvard.edu. FAU - Miettinen, Teemu P AU - Miettinen TP AUID- ORCID: 0000-0002-5975-200X AD - Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. FAU - Skinner, Owen S AU - Skinner OS AD - Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. AD - Department of Systems Biology, Harvard Medical School, Boston, MA, USA. AD - Broad Institute, Cambridge, MA, USA. FAU - Sheedy, Dylan AU - Sheedy D AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AUID- ORCID: 0000-0002-4352-2496 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. AD - Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. FAU - Gapon, Svetlana AU - Gapon S AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. FAU - Hubaud, Alexis AU - Hubaud A AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Manalis, Scott R AU - Manalis SR AUID- ORCID: 0000-0001-5223-9433 AD - Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. AD - Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. FAU - Oldham, William M AU - Oldham WM AUID- ORCID: 0000-0003-3029-4866 AD - Department of Medicine, Harvard Medical School, Boston, MA, USA. AD - Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. FAU - Pourquié, Olivier AU - Pourquié O AUID- ORCID: 0000-0001-5189-1227 AD - Department of Genetics, Harvard Medical School, Boston, MA, USA. pourquie@genetics.med.harvard.edu. AD - Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA. pourquie@genetics.med.harvard.edu. AD - Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA. pourquie@genetics.med.harvard.edu. LA - eng GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - F31 HD100033/HD/NICHD NIH HHS/United States GR - F32 GM133047/GM/NIGMS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States GR - U54 CA217377/CA/NCI NIH HHS/United States GR - R01 HD085121/HD/NICHD NIH HHS/United States PT - Comparative Study PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20230104 PL - England TA - Nature JT - Nature JID - 0410462 RN - 0U46U6E8UK (NAD) RN - EC 1.6.- (NADH oxidase) SB - IM CIN - Nature. 2023 Jan;613(7944):439-440. PMID: 36599994 CIN - Mol Cell. 2023 Mar 16;83(6):824-826. PMID: 36931252 EIN - Nature. 2023 Apr;616(7956):E4. PMID: 36949206 MH - Animals MH - Humans MH - Mice MH - Cell Differentiation MH - *Embryonic Development/physiology MH - NAD/metabolism MH - Oxidation-Reduction MH - Pluripotent Stem Cells/cytology/metabolism MH - Species Specificity MH - In Vitro Techniques MH - Electron Transport MH - Biological Clocks MH - Time Factors MH - *Embryo, Mammalian/cytology/embryology/metabolism MH - Levilactobacillus brevis PMC - PMC9944513 MID - NIHMS1864385 COIS- Competing interests: O.P. is scientific founder of Anagenesis Biotechnologies. S.R.M. is a co-founder of Travera and Affinity Biosensors, which develop technologies relevant to the research presented in this work. All other authors declare no competing interests. EDAT- 2023/01/05 06:00 MHDA- 2023/01/21 06:00 CRDT- 2023/01/04 23:20 PHST- 2021/08/31 00:00 [received] PHST- 2022/11/18 00:00 [accepted] PHST- 2023/01/05 06:00 [pubmed] PHST- 2023/01/21 06:00 [medline] PHST- 2023/01/04 23:20 [entrez] AID - 10.1038/s41586-022-05574-4 [pii] AID - 10.1038/s41586-022-05574-4 [doi] PST - ppublish SO - Nature. 2023 Jan;613(7944):550-557. doi: 10.1038/s41586-022-05574-4. Epub 2023 Jan 4. PMID- 36222651 OWN - NLM STAT- MEDLINE DCOM- 20221026 LR - 20231028 IS - 2050-084X (Electronic) IS - 2050-084X (Linking) VI - 11 DP - 2022 Oct 12 TI - The Na(+)/K(+) pump dominates control of glycolysis in hippocampal dentate granule cells. LID - 10.7554/eLife.81645 [doi] LID - e81645 AB - Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca(2+) influx, but is not activated by feedforward Ca(2+) signaling (Díaz-García et al., 2021a). Since ATP-powered Na(+) and Ca(2+) pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na(+), while intracellular Ca(2+) could only increase NADH production if both forward Na(+)/Ca(2+) exchange and Na(+)/K(+) pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na(+)] increases were reduced >50% by blocking Ca(2+) entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na(+)/K(+) pump, which is strongly potentiated by Na(+) influx through the Na(+)/Ca(2+) exchanger during extrusion of Ca(2+) following stimulation. CI - © 2022, Meyer et al. FAU - Meyer, Dylan J AU - Meyer DJ AUID- ORCID: 0000-0001-8453-3813 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AUID- ORCID: 0000-0002-4352-2496 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Nathwani, Nidhi AU - Nathwani N AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Rahman, Mahia AU - Rahman M AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. LA - eng GR - F32 NS100331/NS/NINDS NIH HHS/United States GR - F32 NS116105/NS/NINDS NIH HHS/United States GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20221012 PL - England TA - Elife JT - eLife JID - 101579614 RN - 0U46U6E8UK (NAD) RN - SY7Q814VUP (Calcium) RN - 9NEZ333N27 (Sodium) RN - 0 (Sodium-Calcium Exchanger) RN - 8L70Q75FXE (Adenosine Triphosphate) RN - 0 (Coloring Agents) SB - IM MH - Animals MH - Mice MH - *NAD/metabolism MH - *Calcium/metabolism MH - Sodium/metabolism MH - Glycolysis/physiology MH - Sodium-Calcium Exchanger/metabolism MH - Hippocampus/metabolism MH - Adenosine Triphosphate/metabolism MH - Coloring Agents PMC - PMC9592084 OTO - NOTNLM OT - Na-Ca exchange OT - Na-K pump OT - cell biology OT - cytosolic NADH OT - glycolysis OT - mouse OT - neurons OT - neuroscience COIS- DM, CD, NN, MR No competing interests declared, GY Reviewing editor, eLife EDAT- 2022/10/13 06:00 MHDA- 2022/10/27 06:00 CRDT- 2022/10/12 09:55 PHST- 2022/07/07 00:00 [received] PHST- 2022/10/11 00:00 [accepted] PHST- 2022/10/13 06:00 [pubmed] PHST- 2022/10/27 06:00 [medline] PHST- 2022/10/12 09:55 [entrez] AID - 81645 [pii] AID - 10.7554/eLife.81645 [doi] PST - epublish SO - Elife. 2022 Oct 12;11:e81645. doi: 10.7554/eLife.81645. PMID- 35614105 OWN - NLM STAT- MEDLINE DCOM- 20220527 LR - 20220716 IS - 2041-1723 (Electronic) IS - 2041-1723 (Linking) VI - 13 IP - 1 DP - 2022 May 25 TI - A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors. PG - 2919 LID - 10.1038/s41467-022-30685-x [doi] LID - 2919 AB - Genetically encoded fluorescent biosensors are powerful tools used to track chemical processes in intact biological systems. However, the development and optimization of biosensors remains a challenging and labor-intensive process, primarily due to technical limitations of methods for screening candidate biosensors. Here we describe a screening modality that combines droplet microfluidics and automated fluorescence imaging to provide an order of magnitude increase in screening throughput. Moreover, unlike current techniques that are limited to screening for a single biosensor feature at a time (e.g. brightness), our method enables evaluation of multiple features (e.g. contrast, affinity, specificity) in parallel. Because biosensor features can covary, this capability is essential for rapid optimization. We use this system to generate a high-performance biosensor for lactate that can be used to quantify intracellular lactate concentrations. This biosensor, named LiLac, constitutes a significant advance in metabolite sensing and demonstrates the power of our screening approach. CI - © 2022. The Author(s). FAU - Koveal, Dorothy AU - Koveal D AUID- ORCID: 0000-0003-2907-6925 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Rosen, Paul C AU - Rosen PC AUID- ORCID: 0000-0001-7414-454X AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. AD - Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. FAU - Meyer, Dylan J AU - Meyer DJ AUID- ORCID: 0000-0001-8453-3813 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AUID- ORCID: 0000-0002-4352-2496 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. AD - Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. FAU - Wang, Yongcheng AU - Wang Y AD - Department of Physics and John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA. AD - Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China. FAU - Cai, Li-Heng AU - Cai LH AUID- ORCID: 0000-0002-6806-0566 AD - Department of Physics and John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA. AD - Department of Materials Science and Engineering, University of Virginia, Charlottesville, VA, USA. FAU - Chou, Peter J AU - Chou PJ AUID- ORCID: 0000-0003-4592-5210 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. AD - Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA. FAU - Weitz, David A AU - Weitz DA AD - Department of Physics and John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. gary_yellen@hms.harvard.edu. LA - eng GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - F32 GM123577/GM/NIGMS NIH HHS/United States GR - P30 EY012196/EY/NEI NIH HHS/United States GR - F31 CA254162/CA/NCI NIH HHS/United States GR - F32 NS116105/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20220525 PL - England TA - Nat Commun JT - Nature communications JID - 101528555 RN - 0 (Coloring Agents) RN - 0 (Lactates) SB - IM MH - *Biosensing Techniques/methods MH - Coloring Agents MH - Lactates MH - Microfluidics PMC - PMC9133083 COIS- The authors declare no competing interests. EDAT- 2022/05/26 06:00 MHDA- 2022/05/28 06:00 CRDT- 2022/05/25 23:18 PHST- 2022/02/23 00:00 [received] PHST- 2022/05/11 00:00 [accepted] PHST- 2022/05/25 23:18 [entrez] PHST- 2022/05/26 06:00 [pubmed] PHST- 2022/05/28 06:00 [medline] AID - 10.1038/s41467-022-30685-x [pii] AID - 30685 [pii] AID - 10.1038/s41467-022-30685-x [doi] PST - epublish SO - Nat Commun. 2022 May 25;13(1):2919. doi: 10.1038/s41467-022-30685-x. PMID- 35493335 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20231117 IS - 2329-423X (Print) IS - 2329-4248 (Electronic) IS - 2329-423X (Linking) VI - 9 IP - Suppl 1 DP - 2022 Jan TI - Neurophotonic tools for microscopic measurements and manipulation: status report. PG - 013001 LID - 10.1117/1.NPh.9.S1.013001 [doi] LID - 013001 AB - Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions. CI - © 2022 The Authors. FAU - Abdelfattah, Ahmed S AU - Abdelfattah AS AD - Brown University, Department of Neuroscience, Providence, Rhode Island, United States. FAU - Ahuja, Sapna AU - Ahuja S AD - University of Pennsylvania, Perelman School of Medicine, Department of Biochemistry and Biophysics, Philadelphia, Pennsylvania, United States. AD - University of Pennsylvania, School of Arts and Sciences, Department of Chemistry, Philadelphia, Pennsylvania, United States. FAU - Akkin, Taner AU - Akkin T AD - University of Minnesota, Department of Biomedical Engineering, Minneapolis, Minnesota, United States. FAU - Allu, Srinivasa Rao AU - Allu SR AD - University of Pennsylvania, Perelman School of Medicine, Department of Biochemistry and Biophysics, Philadelphia, Pennsylvania, United States. AD - University of Pennsylvania, School of Arts and Sciences, Department of Chemistry, Philadelphia, Pennsylvania, United States. FAU - Brake, Joshua AU - Brake J AD - Harvey Mudd College, Department of Engineering, Claremont, California, United States. FAU - Boas, David A AU - Boas DA AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Buckley, Erin M AU - Buckley EM AD - Georgia Institute of Technology and Emory University, Wallace H. Coulter Department of Biomedical Engineering, Atlanta, Georgia, United States. AD - Emory University, Department of Pediatrics, Atlanta, Georgia, United States. FAU - Campbell, Robert E AU - Campbell RE AD - University of Tokyo, Department of Chemistry, Tokyo, Japan. AD - University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada. FAU - Chen, Anderson I AU - Chen AI AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Cheng, Xiaojun AU - Cheng X AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Čižmár, Tomáš AU - Čižmár T AD - Institute of Scientific Instruments of the Czech Academy of Sciences, Brno, Czech Republic. FAU - Costantini, Irene AU - Costantini I AD - University of Florence, European Laboratory for Non-Linear Spectroscopy, Department of Biology, Florence, Italy. AD - National Institute of Optics, National Research Council, Rome, Italy. FAU - De Vittorio, Massimo AU - De Vittorio M AD - Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies, Arnesano, Italy. FAU - Devor, Anna AU - Devor A AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. AD - Massachusetts General Hospital, Harvard Medical School, Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, Massachusetts, United States. FAU - Doran, Patrick R AU - Doran PR AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - El Khatib, Mirna AU - El Khatib M AD - University of Pennsylvania, Perelman School of Medicine, Department of Biochemistry and Biophysics, Philadelphia, Pennsylvania, United States. AD - University of Pennsylvania, School of Arts and Sciences, Department of Chemistry, Philadelphia, Pennsylvania, United States. FAU - Emiliani, Valentina AU - Emiliani V AD - Sorbonne University, INSERM, CNRS, Institut de la Vision, Paris, France. FAU - Fomin-Thunemann, Natalie AU - Fomin-Thunemann N AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Fainman, Yeshaiahu AU - Fainman Y AD - University of California San Diego, Department of Electrical and Computer Engineering, La Jolla, California, United States. FAU - Fernandez-Alfonso, Tomas AU - Fernandez-Alfonso T AD - University College London, Department of Neuroscience, Physiology and Pharmacology, London, United Kingdom. FAU - Ferri, Christopher G L AU - Ferri CGL AD - University of California San Diego, Departments of Neurosciences, La Jolla, California, United States. FAU - Gilad, Ariel AU - Gilad A AD - The Hebrew University of Jerusalem, Institute for Medical Research Israel-Canada, Department of Medical Neurobiology, Faculty of Medicine, Jerusalem, Israel. FAU - Han, Xue AU - Han X AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Harris, Andrew AU - Harris A AD - Weizmann Institute of Science, Department of Brain Sciences, Rehovot, Israel. FAU - Hillman, Elizabeth M C AU - Hillman EMC AD - Columbia University, Zuckerman Mind Brain Behavior Institute, New York, United States. FAU - Hochgeschwender, Ute AU - Hochgeschwender U AD - Central Michigan University, Department of Neuroscience, Mount Pleasant, Michigan, United States. FAU - Holt, Matthew G AU - Holt MG AD - University of Porto, Instituto de Investigação e Inovação em Saúde (i3S), Porto, Portugal. FAU - Ji, Na AU - Ji N AD - University of California Berkeley, Department of Physics, Berkeley, California, United States. FAU - Kılıç, Kıvılcım AU - Kılıç K AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Lake, Evelyn M R AU - Lake EMR AD - Yale School of Medicine, Department of Radiology and Biomedical Imaging, New Haven, Connecticut, United States. FAU - Li, Lei AU - Li L AD - California Institute of Technology, Andrew and Peggy Cherng Department of Medical Engineering, Department of Electrical Engineering, Pasadena, California, United States. FAU - Li, Tianqi AU - Li T AD - University of Minnesota, Department of Biomedical Engineering, Minneapolis, Minnesota, United States. FAU - Mächler, Philipp AU - Mächler P AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Miller, Evan W AU - Miller EW AD - University of California Berkeley, Departments of Chemistry and Molecular & Cell Biology and Helen Wills Neuroscience Institute, Berkeley, California, United States. FAU - Mesquita, Rickson C AU - Mesquita RC AD - University of Campinas, Institute of Physics, Campinas, São Paulo, Brazil. FAU - Nadella, K M Naga Srinivas AU - Nadella KMNS AD - University College London, Department of Neuroscience, Physiology and Pharmacology, London, United Kingdom. FAU - Nägerl, U Valentin AU - Nägerl UV AD - Interdisciplinary Institute for Neuroscience University of Bordeaux & CNRS, Bordeaux, France. FAU - Nasu, Yusuke AU - Nasu Y AD - University of Tokyo, Department of Chemistry, Tokyo, Japan. FAU - Nimmerjahn, Axel AU - Nimmerjahn A AD - Salk Institute for Biological Studies, Waitt Advanced Biophotonics Center, La Jolla, California, United States. FAU - Ondráčková, Petra AU - Ondráčková P AD - Institute of Scientific Instruments of the Czech Academy of Sciences, Brno, Czech Republic. FAU - Pavone, Francesco S AU - Pavone FS AD - National Institute of Optics, National Research Council, Rome, Italy. AD - University of Florence, European Laboratory for Non-Linear Spectroscopy, Department of Physics, Florence, Italy. FAU - Perez Campos, Citlali AU - Perez Campos C AD - Columbia University, Zuckerman Mind Brain Behavior Institute, New York, United States. FAU - Peterka, Darcy S AU - Peterka DS AD - Columbia University, Zuckerman Mind Brain Behavior Institute, New York, United States. FAU - Pisano, Filippo AU - Pisano F AD - Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies, Arnesano, Italy. FAU - Pisanello, Ferruccio AU - Pisanello F AD - Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies, Arnesano, Italy. FAU - Puppo, Francesca AU - Puppo F AD - University of California San Diego, Departments of Neurosciences, La Jolla, California, United States. FAU - Sabatini, Bernardo L AU - Sabatini BL AD - Harvard Medical School, Howard Hughes Medical Institute, Department of Neurobiology, Boston, Massachusetts, United States. FAU - Sadegh, Sanaz AU - Sadegh S AD - University of California San Diego, Departments of Neurosciences, La Jolla, California, United States. FAU - Sakadzic, Sava AU - Sakadzic S AD - Massachusetts General Hospital, Harvard Medical School, Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, Massachusetts, United States. FAU - Shoham, Shy AU - Shoham S AD - New York University Grossman School of Medicine, Tech4Health and Neuroscience Institutes, New York, New York, United States. FAU - Shroff, Sanaya N AU - Shroff SN AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Silver, R Angus AU - Silver RA AD - University College London, Department of Neuroscience, Physiology and Pharmacology, London, United Kingdom. FAU - Sims, Ruth R AU - Sims RR AD - Sorbonne University, INSERM, CNRS, Institut de la Vision, Paris, France. FAU - Smith, Spencer L AU - Smith SL AD - University of California Santa Barbara, Department of Electrical and Computer Engineering, Santa Barbara, California, United States. FAU - Srinivasan, Vivek J AU - Srinivasan VJ AD - New York University Langone Health, Departments of Ophthalmology and Radiology, New York, New York, United States. FAU - Thunemann, Martin AU - Thunemann M AD - Boston University, Department of Biomedical Engineering, Boston, Massachusetts, United States. FAU - Tian, Lei AU - Tian L AD - Boston University, Departments of Electrical Engineering and Biomedical Engineering, Boston, Massachusetts, United States. FAU - Tian, Lin AU - Tian L AD - University of California Davis, Department of Biochemistry and Molecular Medicine, Davis, California, United States. FAU - Troxler, Thomas AU - Troxler T AD - University of Pennsylvania, Perelman School of Medicine, Department of Biochemistry and Biophysics, Philadelphia, Pennsylvania, United States. AD - University of Pennsylvania, School of Arts and Sciences, Department of Chemistry, Philadelphia, Pennsylvania, United States. FAU - Valera, Antoine AU - Valera A AD - University College London, Department of Neuroscience, Physiology and Pharmacology, London, United Kingdom. FAU - Vaziri, Alipasha AU - Vaziri A AD - Rockefeller University, Laboratory of Neurotechnology and Biophysics, New York, New York, United States. AD - The Rockefeller University, The Kavli Neural Systems Institute, New York, New York, United States. FAU - Vinogradov, Sergei A AU - Vinogradov SA AD - University of Pennsylvania, Perelman School of Medicine, Department of Biochemistry and Biophysics, Philadelphia, Pennsylvania, United States. AD - University of Pennsylvania, School of Arts and Sciences, Department of Chemistry, Philadelphia, Pennsylvania, United States. FAU - Vitale, Flavia AU - Vitale F AD - Center for Neuroengineering and Therapeutics, Departments of Neurology, Bioengineering, Physical Medicine and Rehabilitation, Philadelphia, Pennsylvania, United States. FAU - Wang, Lihong V AU - Wang LV AD - California Institute of Technology, Andrew and Peggy Cherng Department of Medical Engineering, Department of Electrical Engineering, Pasadena, California, United States. FAU - Uhlířová, Hana AU - Uhlířová H AD - Institute of Scientific Instruments of the Czech Academy of Sciences, Brno, Czech Republic. FAU - Xu, Chris AU - Xu C AD - Cornell University, School of Applied and Engineering Physics, Ithaca, New York, United States. FAU - Yang, Changhuei AU - Yang C AD - California Institute of Technology, Departments of Electrical Engineering, Bioengineering and Medical Engineering, Pasadena, California, United States. FAU - Yang, Mu-Han AU - Yang MH AD - University of California San Diego, Department of Electrical and Computer Engineering, La Jolla, California, United States. FAU - Yellen, Gary AU - Yellen G AD - Harvard Medical School, Department of Neurobiology, Boston, Massachusetts, United States. FAU - Yizhar, Ofer AU - Yizhar O AD - Weizmann Institute of Science, Department of Brain Sciences, Rehovot, Israel. FAU - Zhao, Yongxin AU - Zhao Y AD - Carnegie Mellon University, Department of Biological Sciences, Pittsburgh, Pennsylvania, United States. LA - eng GR - U01 NS103488/NS/NINDS NIH HHS/United States GR - U01 NS099717/NS/NINDS NIH HHS/United States GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - R01 NS108472/NS/NINDS NIH HHS/United States GR - R01 NS094681/NS/NINDS NIH HHS/United States GR - R01 NS108034/NS/NINDS NIH HHS/United States GR - U01 CA236554/CA/NCI NIH HHS/United States GR - R01 NS109885/NS/NINDS NIH HHS/United States GR - R01 NS115401/NS/NINDS NIH HHS/United States GR - R01 NS098088/NS/NINDS NIH HHS/United States GR - U19 NS107613/NS/NINDS NIH HHS/United States GR - R21 EY030016/EY/NEI NIH HHS/United States GR - U19 NS123719/NS/NINDS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States GR - R01 NS117756/NS/NINDS NIH HHS/United States GR - UF1 NS108213/NS/NINDS NIH HHS/United States GR - U01 NS113273/NS/NINDS NIH HHS/United States GR - DP2 MH129956/MH/NIMH NIH HHS/United States GR - R44 MH117430/MH/NIMH NIH HHS/United States GR - U19 NS123717/NS/NINDS NIH HHS/United States GR - UF1 NS108177/NS/NINDS NIH HHS/United States GR - U24 EB028941/EB/NIBIB NIH HHS/United States GR - RF1 NS121095/NS/NINDS NIH HHS/United States GR - U01 NS118300/NS/NINDS NIH HHS/United States GR - F31 NS118949/NS/NINDS NIH HHS/United States GR - U01 NS094296/NS/NINDS NIH HHS/United States GR - R01 NS091230/NS/NINDS NIH HHS/United States GR - K25 HL145092/HL/NHLBI NIH HHS/United States GR - R01 NS120832/NS/NINDS NIH HHS/United States GR - R01 NS121219/NS/NINDS NIH HHS/United States GR - R01 EY031469/EY/NEI NIH HHS/United States GR - F31 NS115421/NS/NINDS NIH HHS/United States GR - R01 EB029747/EB/NIBIB NIH HHS/United States GR - RF1 NS110501/NS/NINDS NIH HHS/United States GR - RF1 NS113251/NS/NINDS NIH HHS/United States GR - R01 DA050159/DA/NIDA NIH HHS/United States GR - U19 NS112959/NS/NINDS NIH HHS/United States GR - U01 EB029823/EB/NIBIB NIH HHS/United States PT - Journal Article DEP - 20220427 PL - United States TA - Neurophotonics JT - Neurophotonics JID - 101632875 PMC - PMC9047450 OTO - NOTNLM OT - blood flow OT - fluorescence OT - label free OT - molecular sensors OT - multimodal OT - optical imaging OT - optogenetics EDAT- 2022/05/03 06:00 MHDA- 2022/05/03 06:01 CRDT- 2022/05/02 06:18 PHST- 2022/05/02 06:18 [entrez] PHST- 2022/05/03 06:00 [pubmed] PHST- 2022/05/03 06:01 [medline] AID - 22-0308 [pii] AID - 10.1117/1.NPh.9.S1.013001 [doi] PST - ppublish SO - Neurophotonics. 2022 Jan;9(Suppl 1):013001. doi: 10.1117/1.NPh.9.S1.013001. Epub 2022 Apr 27. PMID- 35087918 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20230704 IS - 2331-8325 (Electronic) IS - 2331-8325 (Linking) VI - 11 IP - 24 DP - 2021 Dec 20 TI - Delivery of AAV for Expression of Fluorescent Biosensors in Juvenile Mouse Hippocampus. PG - e4259 LID - 10.21769/BioProtoc.4259 [doi] LID - e4259 AB - Genetically encoded fluorescent biosensors are versatile tools for studying brain metabolism and function in live tissue. The genetic information for these biosensors can be delivered into the brain by stereotaxic injection of engineered adeno-associated viruses (AAVs), which can selectively target different cell types depending on the capsid serotype and/or the viral promoter. Here, we describe a protocol for intracranial injections of two viral vectors encoding the metabolic biosensor Peredox and the calcium biosensor RCaMP1h. When combined with 2-photon microscopy and fluorescence lifetime imaging, this protocol allows the simultaneous quantitative assessment of changes in the cytosolic NADH/NAD(+) ratio and the intracellular Ca(2+) levels in individual dentate granule cells from acute hippocampal slices. Graphic abstract: Workflow diagram for biosensor expression in the mouse hippocampus using intracranial injections of adeno-associated viruses. CI - Copyright © The Authors; exclusive licensee Bio-protocol LLC. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Nathwani, Nidhi AU - Nathwani N AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Martínez-François, Juan Ramón AU - Martínez-François JR AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. LA - eng GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - F32 NS100331/NS/NINDS NIH HHS/United States GR - DP1 EB016986/EB/NIBIB NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article DEP - 20211220 PL - United States TA - Bio Protoc JT - Bio-protocol JID - 101635102 PMC - PMC8720514 OTO - NOTNLM OT - Acute brain slices OT - Adeno-associated viruses OT - Dentate granule cells OT - Genetically-encoded fluorescent biosensors OT - Hippocampus OT - Intracranial injection COIS- Competing interestsThe authors do not have any competing interest to declare. EDAT- 2022/01/29 06:00 MHDA- 2022/01/29 06:01 CRDT- 2022/01/28 05:55 PHST- 2021/07/23 00:00 [received] PHST- 2021/09/22 00:00 [revised] PHST- 2021/09/28 00:00 [accepted] PHST- 2022/01/28 05:55 [entrez] PHST- 2022/01/29 06:00 [pubmed] PHST- 2022/01/29 06:01 [medline] AID - e4259 [pii] AID - 4259 [pii] AID - 10.21769/BioProtoc.4259 [doi] PST - epublish SO - Bio Protoc. 2021 Dec 20;11(24):e4259. doi: 10.21769/BioProtoc.4259. eCollection 2021 Dec 20. PMID- 34247456 OWN - NLM STAT- MEDLINE DCOM- 20220124 LR - 20231107 IS - 2328-9503 (Electronic) IS - 2328-9503 (Linking) VI - 8 IP - 8 DP - 2021 Aug TI - Metabolism-based therapies for epilepsy: new directions for future cures. PG - 1730-1737 LID - 10.1002/acn3.51423 [doi] AB - OBJECTIVE: Thousands of years after dietary therapy was proposed to treat seizures, how alterations in metabolism relates to epilepsy remains unclear, and metabolism-based therapies are not always effective. METHODS: We consider the state of the science in metabolism-based therapies for epilepsy across the research lifecycle from basic to translational to clinical studies. RESULTS: This analysis creates a conceptual framework for creative, rigorous, and transparent research to benefit people with epilepsy through the understanding and modification of metabolism. INTERPRETATION: Despite intensive past efforts to evaluate metabolism-based therapies for epilepsy, distinct ways of framing a problem offer the chance to engage different mindsets and new (or newly applied) technologies. A comprehensive, creative, and inclusive problem-directed research agenda is needed, with a renewed and stringent adherence to rigor and transparency across all levels of investigation. CI - © 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association. This article has been contributed to by US Government employees and their work is in the public domain in the USA. FAU - Cervenka, Mackenzie AU - Cervenka M AD - Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. FAU - Pascual, Juan M AU - Pascual JM AD - Department of Neurology, University of Texas Southwestern, Dallas, Texas, USA. FAU - Rho, Jong M AU - Rho JM AUID- ORCID: 0000-0001-9886-9924 AD - Departments of Neurosciences and Pediatrics, University of California, San Diego, California, USA. FAU - Thiele, Elizabeth AU - Thiele E AD - Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. FAU - Whittemore, Vicky AU - Whittemore V AD - National Institute of Neurological Disorders and Stroke, National Institutes of Health, Rockville, Maryland, USA. FAU - Hartman, Adam L AU - Hartman AL AUID- ORCID: 0000-0001-5672-3409 AD - National Institute of Neurological Disorders and Stroke, National Institutes of Health, Rockville, Maryland, USA. LA - eng GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - R01 NS094257/NS/NINDS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States GR - R21 NS104513/NS/NINDS NIH HHS/United States GR - R01 NS102588/NS/NINDS NIH HHS/United States GR - R37 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Review DEP - 20210711 PL - United States TA - Ann Clin Transl Neurol JT - Annals of clinical and translational neurology JID - 101623278 SB - IM MH - Epilepsy/*metabolism/*therapy MH - Humans PMC - PMC8351378 COIS- Drs Pascual, Yellen, Thiele, Whittemore, and Hartman report no conflicts of interest. Dr Rho serves as a consultant for Aquestive Therapeutics, Cerecin Ltd., Biocodex, and Cypralis Ltd. Dr. Cervenka has received grants from Vitaflo International Ltd, honoraria from Nutricia and Vitaflo International Ltd, royalties from Demos Health/Springer Publishing Company, and consulting fees for Nutricia. EDAT- 2021/07/12 06:00 MHDA- 2022/01/27 06:00 CRDT- 2021/07/11 21:07 PHST- 2021/05/14 00:00 [received] PHST- 2021/06/28 00:00 [accepted] PHST- 2021/07/12 06:00 [pubmed] PHST- 2022/01/27 06:00 [medline] PHST- 2021/07/11 21:07 [entrez] AID - ACN351423 [pii] AID - 10.1002/acn3.51423 [doi] PST - ppublish SO - Ann Clin Transl Neurol. 2021 Aug;8(8):1730-1737. doi: 10.1002/acn3.51423. Epub 2021 Jul 11. PMID- 33555254 OWN - NLM STAT- MEDLINE DCOM- 20220208 LR - 20220208 IS - 2050-084X (Electronic) IS - 2050-084X (Linking) VI - 10 DP - 2021 Feb 8 TI - The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle. LID - 10.7554/eLife.64821 [doi] LID - e64821 AB - When neurons engage in intense periods of activity, the consequent increase in energy demand can be met by the coordinated activation of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, the trigger for glycolytic activation is unknown and the role for Ca(2+) in the mitochondrial responses has been debated. Using genetically encoded fluorescent biosensors and NAD(P)H autofluorescence imaging in acute hippocampal slices, here we find that Ca(2+) uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca(2+) activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca(2+)/calmodulin signaling pathway, or AMPK, in mediating the rise in glycolytic NADH in response to acute stimulation. Aerobic glycolysis in neurons is triggered mainly by the energy demand resulting from either Na(+) or Ca(2+) extrusion, and in mouse dentate granule cells, Ca(2+) creates the majority of this demand. CI - © 2021, Díaz-García et al. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AUID- ORCID: 0000-0002-4352-2496 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Meyer, Dylan J AU - Meyer DJ AUID- ORCID: 0000-0001-8453-3813 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Nathwani, Nidhi AU - Nathwani N AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Rahman, Mahia AU - Rahman M AUID- ORCID: 0000-0001-6870-4221 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Martínez-François, Juan Ramón AU - Martínez-François JR AUID- ORCID: 0000-0002-1035-2574 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. LA - eng GR - F32 NS100331/NS/NINDS NIH HHS/United States GR - DP1 EB016986/EB/NIBIB NIH HHS/United States GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - F32 NS116105/NS/NINDS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20210208 PL - England TA - Elife JT - eLife JID - 101579614 RN - 0U46U6E8UK (NAD) RN - 53-59-8 (NADP) RN - 9NEZ333N27 (Sodium) RN - SY7Q814VUP (Calcium) SB - IM MH - Animals MH - Brain/cytology/metabolism MH - Calcium/*metabolism MH - *Citric Acid Cycle MH - Cytosol/metabolism MH - Energy Metabolism MH - Female MH - Glycolysis MH - Male MH - Mice MH - Mice, Inbred C57BL MH - Mitochondria/metabolism MH - NAD/metabolism MH - NADP/metabolism MH - Neurons/*metabolism MH - Oxidative Phosphorylation MH - Sodium/metabolism PMC - PMC7870136 OTO - NOTNLM OT - brain metabolism OT - mitochondrial calcium OT - mitochondrial calcium uniporter OT - mouse OT - neuronal glycolysis OT - neuroscience COIS- CD, DM, NN, MR, JM No competing interests declared, GY Reviewing editor, eLife EDAT- 2021/02/09 06:00 MHDA- 2022/02/09 06:00 CRDT- 2021/02/08 12:09 PHST- 2020/11/12 00:00 [received] PHST- 2021/01/26 00:00 [accepted] PHST- 2021/02/08 12:09 [entrez] PHST- 2021/02/09 06:00 [pubmed] PHST- 2022/02/09 06:00 [medline] AID - 64821 [pii] AID - 10.7554/eLife.64821 [doi] PST - epublish SO - Elife. 2021 Feb 8;10:e64821. doi: 10.7554/eLife.64821. PMID- 32559637 OWN - NLM STAT- MEDLINE DCOM- 20201109 LR - 20210802 IS - 1873-6882 (Electronic) IS - 0959-4388 (Print) IS - 0959-4388 (Linking) VI - 63 DP - 2020 Aug TI - Fluorescent Biosensors for Neuronal Metabolism and the Challenges of Quantitation. PG - 111-121 LID - S0959-4388(20)30049-0 [pii] LID - 10.1016/j.conb.2020.02.011 [doi] AB - Over the past decade, genetically encoded fluorescent biosensors that report metabolic changes have become valuable tools for understanding brain metabolism. These sensors have been targeted to specific brain regions and cell types in different organisms to track multiple metabolic processes at single cell (and subcellular) resolution. Here, we review genetically encoded biosensors used to study metabolism in the brain. We particularly focus on the principles needed to use these sensors quantitatively while avoiding false inferences from variations in sensor fluorescence that arise from differences in expression level or environmental influences such as pH or temperature. CI - Copyright © 2020. Published by Elsevier Ltd. FAU - Koveal, Dorothy AU - Koveal D AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. Electronic address: gary_yellen@hms.harvard.edu. LA - eng GR - F32 GM123577/GM/NIGMS NIH HHS/United States GR - F32 NS100331/NS/NINDS NIH HHS/United States GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Review DEP - 20200616 PL - England TA - Curr Opin Neurobiol JT - Current opinion in neurobiology JID - 9111376 SB - IM MH - *Biosensing Techniques MH - *Fluorescence Resonance Energy Transfer MH - Neurons PMC - PMC7646541 MID - NIHMS1635082 EDAT- 2020/06/20 06:00 MHDA- 2020/11/11 06:00 CRDT- 2020/06/20 06:00 PHST- 2020/02/13 00:00 [received] PHST- 2020/02/18 00:00 [revised] PHST- 2020/02/25 00:00 [accepted] PHST- 2020/06/20 06:00 [pubmed] PHST- 2020/11/11 06:00 [medline] PHST- 2020/06/20 06:00 [entrez] AID - S0959-4388(20)30049-0 [pii] AID - 10.1016/j.conb.2020.02.011 [doi] PST - ppublish SO - Curr Opin Neurobiol. 2020 Aug;63:111-121. doi: 10.1016/j.conb.2020.02.011. Epub 2020 Jun 16. PMID- 32461692 OWN - NLM STAT- MEDLINE DCOM- 20200723 LR - 20230120 IS - 1476-4687 (Electronic) IS - 0028-0836 (Print) IS - 0028-0836 (Linking) VI - 583 IP - 7814 DP - 2020 Jul TI - Hepatic NADH reductive stress underlies common variation in metabolic traits. PG - 122-126 LID - 10.1038/s41586-020-2337-2 [doi] AB - The cellular NADH/NAD(+) ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX(1), a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD(+) ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD(+) ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance(2), insulin resistance(3) and mitochondrial disease(4), and are associated with a common genetic variant in GCKR(5), which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD(+) ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'. FAU - Goodman, Russell P AU - Goodman RP AD - Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. AD - Liver Center, Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, USA. FAU - Markhard, Andrew L AU - Markhard AL AD - Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. FAU - Shah, Hardik AU - Shah H AUID- ORCID: 0000-0001-8408-5686 AD - Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. FAU - Sharma, Rohit AU - Sharma R AD - Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. FAU - Skinner, Owen S AU - Skinner OS AD - Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. FAU - Clish, Clary B AU - Clish CB AUID- ORCID: 0000-0001-8259-9245 AD - Broad Institute, Cambridge, MA, USA. FAU - Deik, Amy AU - Deik A AD - Broad Institute, Cambridge, MA, USA. FAU - Patgiri, Anupam AU - Patgiri A AD - Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. FAU - Hsu, Yu-Han H AU - Hsu YH AD - Broad Institute, Cambridge, MA, USA. AD - Departments of Pediatrics and Genetics, Harvard Medical School, Boston, MA, USA. AD - Division of Endocrinology and Center for Basic and Translational Obesity Research, Boston Children's Hospital, Boston, MA, USA. FAU - Masia, Ricard AU - Masia R AD - Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Noh, Hye Lim AU - Noh HL AD - Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. FAU - Suk, Sujin AU - Suk S AD - Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. FAU - Goldberger, Olga AU - Goldberger O AD - Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. FAU - Hirschhorn, Joel N AU - Hirschhorn JN AD - Broad Institute, Cambridge, MA, USA. AD - Departments of Pediatrics and Genetics, Harvard Medical School, Boston, MA, USA. AD - Division of Endocrinology and Center for Basic and Translational Obesity Research, Boston Children's Hospital, Boston, MA, USA. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Kim, Jason K AU - Kim JK AD - Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. AD - Division of Endocrinology, Metabolism, and Diabetes, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA. FAU - Mootha, Vamsi K AU - Mootha VK AUID- ORCID: 0000-0001-9924-642X AD - Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. vamsi@hms.harvard.edu. AD - Broad Institute, Cambridge, MA, USA. vamsi@hms.harvard.edu. AD - Department of Systems Biology, Harvard Medical School, Boston, MA, USA. vamsi@hms.harvard.edu. LA - eng GR - R01 GM099683/GM/NIGMS NIH HHS/United States GR - P30 DK040561/DK/NIDDK NIH HHS/United States GR - 5U2C-DK093000/NH/NIH HHS/United States GR - R35GM122455/NH/NIH HHS/United States GR - K08DK1158811/NH/NIH HHS/United States GR - R35 GM122455/GM/NIGMS NIH HHS/United States GR - T32 DK110919/DK/NIDDK NIH HHS/United States GR - TR01GM099683/NH/NIH HHS/United States GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - K08 DK115881/DK/NIDDK NIH HHS/United States GR - U2C DK093000/DK/NIDDK NIH HHS/United States GR - F32 GM133047/GM/NIGMS NIH HHS/United States GR - HHMI/Howard Hughes Medical Institute/United States GR - F32 DK111132/DK/NIDDK NIH HHS/United States GR - T32 HG002295/HG/NHGRI NIH HHS/United States GR - R01 DK075787/DK/NIDDK NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20200527 PL - England TA - Nature JT - Nature JID - 0410462 RN - 0 (Adaptor Proteins, Signal Transducing) RN - 0 (GCKR protein, human) RN - 0 (Multienzyme Complexes) RN - 0 (Triglycerides) RN - 0 (fibroblast growth factor 21) RN - 0U46U6E8UK (NAD) RN - 62031-54-3 (Fibroblast Growth Factors) RN - EC 1.6.- (NADH oxidase) RN - EC 1.6.- (NADH, NADPH Oxidoreductases) SB - IM MH - Adaptor Proteins, Signal Transducing/genetics MH - Animals MH - Cytosol/metabolism MH - Disease Models, Animal MH - Fibroblast Growth Factors/blood MH - Genetic Variation MH - Glucose Tolerance Test MH - Humans MH - Insulin Resistance MH - Levilactobacillus brevis/enzymology/genetics MH - Liver/*metabolism MH - Male MH - Mice MH - Multienzyme Complexes/genetics/metabolism MH - NAD/*metabolism MH - NADH, NADPH Oxidoreductases/genetics/metabolism MH - Oxidation-Reduction MH - *Stress, Physiological MH - Triglycerides/blood PMC - PMC7536642 MID - NIHMS1575436 COIS- Competing Financial Interests: V.K.M. is an inventor on a patent PCT/US2016/045015 filed by Massachusetts General Hospital on the use of the LbNOX technology as protein prosthesis for mitochondrial diseases or conditions. V.K.M. and R.P.G are inventors on a patent provisionally filed by Massachusetts General Hospital on modulating hepatic reductive stress with chemicals. EDAT- 2020/05/29 06:00 MHDA- 2020/07/24 06:00 CRDT- 2020/05/29 06:00 PHST- 2019/02/22 00:00 [received] PHST- 2020/03/11 00:00 [accepted] PHST- 2020/05/29 06:00 [pubmed] PHST- 2020/07/24 06:00 [medline] PHST- 2020/05/29 06:00 [entrez] AID - 10.1038/s41586-020-2337-2 [pii] AID - 10.1038/s41586-020-2337-2 [doi] PST - ppublish SO - Nature. 2020 Jul;583(7814):122-126. doi: 10.1038/s41586-020-2337-2. Epub 2020 May 27. PMID- 31106909 OWN - NLM STAT- MEDLINE DCOM- 20200728 LR - 20231012 IS - 1097-4547 (Electronic) IS - 0360-4012 (Print) IS - 0360-4012 (Linking) VI - 97 IP - 8 DP - 2019 Aug TI - Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor. PG - 946-960 LID - 10.1002/jnr.24433 [doi] AB - Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4-2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7-2.5 mM in cortical neurons from awake mice. CI - © 2019 Wiley Periodicals, Inc. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AUID- ORCID: 0000-0002-4352-2496 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. FAU - Lahmann, Carolina AU - Lahmann C AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. FAU - Martínez-François, Juan Ramón AU - Martínez-François JR AUID- ORCID: 0000-0002-1035-2574 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. FAU - Li, Binsen AU - Li B AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. FAU - Koveal, Dorothy AU - Koveal D AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. FAU - Nathwani, Nidhi AU - Nathwani N AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. FAU - Rahman, Mahia AU - Rahman M AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. FAU - Keller, Jacob P AU - Keller JP AD - Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia. FAU - Marvin, Jonathan S AU - Marvin JS AUID- ORCID: 0000-0003-2294-4515 AD - Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia. FAU - Looger, Loren L AU - Looger LL AD - Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. LA - eng GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - F32 GM123577/GM/NIGMS NIH HHS/United States GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - F32 NS093784/NS/NINDS NIH HHS/United States GR - F32 NS100331/NS/NINDS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20190520 PL - United States TA - J Neurosci Res JT - Journal of neuroscience research JID - 7600111 RN - 0 (Luminescent Proteins) RN - IY9XDZ35W2 (Glucose) SB - IM MH - Animals MH - Biosensing Techniques/instrumentation/*methods MH - Female MH - Genetic Vectors MH - Glucose/*metabolism MH - HEK293 Cells MH - Hippocampus/*metabolism MH - Humans MH - Luminescent Proteins/genetics/metabolism MH - Male MH - Mice, Inbred C57BL MH - Neurons/*metabolism MH - Spectrometry, Fluorescence/*methods MH - Thermus thermophilus/genetics PMC - PMC6565483 MID - NIHMS1526614 OTO - NOTNLM OT - energy metabolism OT - fluorescent biosensor OT - glucose metabolism COIS- CONFLICT OF INTEREST The authors report no conflicts of interest. EDAT- 2019/05/21 06:00 MHDA- 2020/07/29 06:00 CRDT- 2019/05/21 06:00 PHST- 2019/01/24 00:00 [received] PHST- 2019/04/08 00:00 [revised] PHST- 2019/04/08 00:00 [accepted] PHST- 2019/05/21 06:00 [pubmed] PHST- 2020/07/29 06:00 [medline] PHST- 2019/05/21 06:00 [entrez] AID - 10.1002/jnr.24433 [doi] PST - ppublish SO - J Neurosci Res. 2019 Aug;97(8):946-960. doi: 10.1002/jnr.24433. Epub 2019 May 20. PMID- 30575090 OWN - NLM STAT- MEDLINE DCOM- 20200728 LR - 20200728 IS - 1097-4547 (Electronic) IS - 0360-4012 (Print) IS - 0360-4012 (Linking) VI - 97 IP - 8 DP - 2019 Aug TI - Neurons rely on glucose rather than astrocytic lactate during stimulation. PG - 883-889 LID - 10.1002/jnr.24374 [doi] AB - Brain metabolism increases during stimulation, but this increase does not affect all energy metabolism equally. Briefly after stimulation, there is a local increase in cerebral blood flow and in glucose uptake, but a smaller increase in oxygen uptake. This indicates that temporarily the rate of glycolysis is faster than the rate of oxidative metabolism, with a corresponding temporary increase in lactate production. This minireview discusses the long-standing controversy about which cell type, neurons or astrocytes, are involved in this increased aerobic glycolysis. Recent biosensor studies measuring metabolic changes in neurons, in acute brain slices or in vivo, are placed in the context of other data bearing on this question. The most direct measurements indicate that, although both neurons and astrocytes may increase glycolysis after stimulation, neurons do not rely on import of astrocytic-produced lactate, and instead they increase their own glycolytic rate and become net exporters of lactate. This temporary increase in neuronal glycolysis may provide rapid energy to meet the acute energy demands of neurons. CI - © 2018 Wiley Periodicals, Inc. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AUID- ORCID: 0000-0002-4352-2496 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. LA - eng GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - F32 NS100331/NS/NINDS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Review DEP - 20181221 PL - United States TA - J Neurosci Res JT - Journal of neuroscience research JID - 7600111 RN - 33X04XA5AT (Lactic Acid) RN - IY9XDZ35W2 (Glucose) SB - IM MH - Animals MH - Astrocytes/*metabolism MH - Brain/*metabolism MH - Energy Metabolism MH - Glucose/*metabolism MH - Glycolysis MH - Humans MH - Lactic Acid/*metabolism MH - Neurons/*metabolism PMC - PMC6565458 MID - NIHMS1515744 COIS- AUTHOR STATEMENTS The authors report no conflicts of interest. Both authors contributed to the design and preparation of this review. Writing – Original Draft, C.M.D-G. and G.Y.; Writing – Review and Editing, C.M.D-G. and G.Y.; Visualization, G.Y.; Funding Acquisition, C.M.D-G. and G.Y. EDAT- 2018/12/24 06:00 MHDA- 2020/07/29 06:00 CRDT- 2018/12/22 06:00 PHST- 2018/10/31 00:00 [received] PHST- 2018/11/29 00:00 [revised] PHST- 2018/12/03 00:00 [accepted] PHST- 2018/12/24 06:00 [pubmed] PHST- 2020/07/29 06:00 [medline] PHST- 2018/12/22 06:00 [entrez] AID - 10.1002/jnr.24374 [doi] PST - ppublish SO - J Neurosci Res. 2019 Aug;97(8):883-889. doi: 10.1002/jnr.24374. Epub 2018 Dec 21. PMID- 29752396 OWN - NLM STAT- MEDLINE DCOM- 20190520 LR - 20190520 IS - 1540-8140 (Electronic) IS - 0021-9525 (Print) IS - 0021-9525 (Linking) VI - 217 IP - 7 DP - 2018 Jul 2 TI - Fueling thought: Management of glycolysis and oxidative phosphorylation in neuronal metabolism. PG - 2235-2246 LID - 10.1083/jcb.201803152 [doi] AB - The brain's energy demands are remarkable both in their intensity and in their moment-to-moment dynamic range. This perspective considers the evidence for Warburg-like aerobic glycolysis during the transient metabolic response of the brain to acute activation, and it particularly addresses the cellular mechanisms that underlie this metabolic response. The temporary uncoupling between glycolysis and oxidative phosphorylation led to the proposal of an astrocyte-to-neuron lactate shuttle whereby during stimulation, lactate produced by increased glycolysis in astrocytes is taken up by neurons as their primary energy source. However, direct evidence for this idea is lacking, and evidence rather supports that neurons have the capacity to increase their own glycolysis in response to stimulation; furthermore, neurons may export rather than import lactate in response to stimulation. The possible cellular mechanisms for invoking metabolic resupply of energy in neurons are also discussed, in particular the roles of feedback signaling via adenosine diphosphate and feedforward signaling by calcium ions. CI - © 2018 Yellen. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, MA. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - R01 GM124038/GM/NIGMS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Review DEP - 20180511 PL - United States TA - J Cell Biol JT - The Journal of cell biology JID - 0375356 RN - 33X04XA5AT (Lactic Acid) RN - 61D2G4IYVH (Adenosine Diphosphate) RN - IY9XDZ35W2 (Glucose) RN - SY7Q814VUP (Calcium) SB - IM MH - Adenosine Diphosphate/metabolism MH - Animals MH - Astrocytes/metabolism MH - Brain/*metabolism MH - Calcium/metabolism MH - Energy Metabolism/*genetics MH - Glucose/metabolism MH - Glycolysis/genetics MH - Humans MH - Lactic Acid/metabolism MH - Neurons/*metabolism MH - *Oxidative Phosphorylation PMC - PMC6028533 EDAT- 2018/05/13 06:00 MHDA- 2019/05/21 06:00 CRDT- 2018/05/13 06:00 PHST- 2018/03/26 00:00 [received] PHST- 2018/04/30 00:00 [revised] PHST- 2018/05/02 00:00 [accepted] PHST- 2018/05/13 06:00 [pubmed] PHST- 2019/05/21 06:00 [medline] PHST- 2018/05/13 06:00 [entrez] AID - jcb.201803152 [pii] AID - 201803152 [pii] AID - 10.1083/jcb.201803152 [doi] PST - ppublish SO - J Cell Biol. 2018 Jul 2;217(7):2235-2246. doi: 10.1083/jcb.201803152. Epub 2018 May 11. PMID- 29368690 OWN - NLM STAT- MEDLINE DCOM- 20180830 LR - 20211001 IS - 2050-084X (Electronic) IS - 2050-084X (Linking) VI - 7 DP - 2018 Jan 25 TI - BAD and K(ATP) channels regulate neuron excitability and epileptiform activity. LID - 10.7554/eLife.32721 [doi] LID - e32721 AB - Brain metabolism can profoundly influence neuronal excitability. Mice with genetic deletion or alteration of Bad (BCL-2 agonist of cell death) exhibit altered brain-cell fuel metabolism, accompanied by resistance to acutely induced epileptic seizures; this seizure protection is mediated by ATP-sensitive potassium (K(ATP)) channels. Here we investigated the effect of BAD manipulation on K(ATP) channel activity and excitability in acute brain slices. We found that BAD's influence on neuronal K(ATP) channels was cell-autonomous and directly affected dentate granule neuron (DGN) excitability. To investigate the role of neuronal K(ATP) channels in the anticonvulsant effects of BAD, we imaged calcium during picrotoxin-induced epileptiform activity in entorhinal-hippocampal slices. BAD knockout reduced epileptiform activity, and this effect was lost upon knockout or pharmacological inhibition of K(ATP) channels. Targeted BAD knockout in DGNs alone was sufficient for the antiseizure effect in slices, consistent with a 'dentate gate' function that is reinforced by increased K(ATP) channel activity. CI - © 2018, Martínez-François et al. FAU - Martínez-François, Juan Ramón AU - Martínez-François JR AUID- ORCID: 0000-0002-1035-2574 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Fernández-Agüera, María Carmen AU - Fernández-Agüera MC AUID- ORCID: 0000-0002-0769-213X AD - Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, United States. FAU - Nathwani, Nidhi AU - Nathwani N AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Lahmann, Carolina AU - Lahmann C AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Burnham, Veronica L AU - Burnham VL AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Danial, Nika N AU - Danial NN AD - Department of Neurobiology, Harvard Medical School, Boston, United States. AD - Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, United States. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. LA - eng GR - P30 NS072030/NS/NINDS NIH HHS/United States GR - R01 NS083844/NH/NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - P30 EY012196/EY/NEI NIH HHS/United States GR - F32 NS093784/NS/NINDS NIH HHS/United States GR - R01 NS055031/NH/NIH HHS/United States GR - 473-2016/European Molecular Biology Organization/International GR - R01 NS083844/NS/NINDS NIH HHS/United States GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States GR - DP1 EB016985/NH/NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20180125 PL - England TA - Elife JT - eLife JID - 101579614 RN - 0 (Bad protein, mouse) RN - 0 (KATP Channels) RN - 0 (bcl-Associated Death Protein) SB - IM CIN - Epilepsy Curr. 2018 Sep-Oct;18(5):332-333. PMID: 30464738 MH - Animals MH - Entorhinal Cortex/*physiology MH - KATP Channels/*metabolism MH - Mice MH - Mice, Knockout MH - Neurons/*physiology MH - Seizures/*physiopathology MH - bcl-Associated Death Protein/genetics/*metabolism PMC - PMC5785210 OTO - NOTNLM OT - Brain slice OT - Calcium imaging OT - Epilepsy OT - Metabolic seizure resistance OT - mouse OT - neuroscience COIS- JM, MF, NN, CL, VB, ND, GY No competing interests declared EDAT- 2018/01/26 06:00 MHDA- 2018/08/31 06:00 CRDT- 2018/01/26 06:00 PHST- 2017/10/11 00:00 [received] PHST- 2018/01/12 00:00 [accepted] PHST- 2018/01/26 06:00 [entrez] PHST- 2018/01/26 06:00 [pubmed] PHST- 2018/08/31 06:00 [medline] AID - 32721 [pii] AID - 10.7554/eLife.32721 [doi] PST - epublish SO - Elife. 2018 Jan 25;7:e32721. doi: 10.7554/eLife.32721. PMID- 29239720 OWN - NLM STAT- MEDLINE DCOM- 20180718 LR - 20190610 IS - 2050-084X (Electronic) IS - 2050-084X (Linking) VI - 6 DP - 2017 Dec 14 TI - Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells. LID - 10.7554/eLife.27293 [doi] LID - e27293 AB - Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD(+) redox. Across various conditions, including glycolytic or mitochondrial inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including both stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations that indicate a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD(+) redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells. FAU - Hung, Yin P AU - Hung YP AUID- ORCID: 0000-0002-8568-1591 AD - Department of Cell Biology, Harvard Medical School, Boston, United States. AD - Department of Neurobiology, Harvard Medical School, Boston, United States. AD - Department of Pathology, Brigham and Women's Hospital, Boston, United States. FAU - Teragawa, Carolyn AU - Teragawa C AD - Department of Molecular and Cellular Biology, University of California, Davis, United States. FAU - Kosaisawe, Nont AU - Kosaisawe N AD - Department of Molecular and Cellular Biology, University of California, Davis, United States. FAU - Gillies, Taryn E AU - Gillies TE AD - Department of Molecular and Cellular Biology, University of California, Davis, United States. FAU - Pargett, Michael AU - Pargett M AD - Department of Molecular and Cellular Biology, University of California, Davis, United States. FAU - Minguet, Marta AU - Minguet M AD - Department of Molecular and Cellular Biology, University of California, Davis, United States. FAU - Distor, Kevin AU - Distor K AD - Department of Molecular and Cellular Biology, University of California, Davis, United States. FAU - Rocha-Gregg, Briana L AU - Rocha-Gregg BL AD - Department of Molecular and Cellular Biology, University of California, Davis, United States. FAU - Coloff, Jonathan L AU - Coloff JL AD - Department of Cell Biology, Harvard Medical School, Boston, United States. FAU - Keibler, Mark A AU - Keibler MA AD - Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, United States. FAU - Stephanopoulos, Gregory AU - Stephanopoulos G AD - Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, United States. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Brugge, Joan S AU - Brugge JS AD - Department of Cell Biology, Harvard Medical School, Boston, United States. FAU - Albeck, John G AU - Albeck JG AUID- ORCID: 0000-0003-2688-8653 AD - Department of Molecular and Cellular Biology, University of California, Davis, United States. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - R01 CA105134/CA/NCI NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - T32 GM007377/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20171214 PL - England TA - Elife JT - eLife JID - 101579614 RN - 0U46U6E8UK (NAD) RN - EC 2.7.1.- (Phosphatidylinositol 3-Kinases) RN - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt) RN - EC 2.7.11.31 (AMP-Activated Protein Kinases) SB - IM MH - AMP-Activated Protein Kinases/analysis MH - Cell Line MH - Cell Proliferation MH - *Energy Metabolism MH - Epithelial Cells/*metabolism MH - *Gene Expression Regulation MH - *Glycolysis MH - Humans MH - NAD/analysis MH - Phosphatidylinositol 3-Kinases/*analysis MH - Proto-Oncogene Proteins c-akt/*analysis PMC - PMC5730373 OTO - NOTNLM OT - AMPK OT - Akt OT - biochemistry OT - cell biology OT - glycolysis OT - homeostasis OT - human OT - primary metabolism OT - single-cell COIS- No competing interests declared. EDAT- 2017/12/15 06:00 MHDA- 2018/07/19 06:00 CRDT- 2017/12/15 06:00 PHST- 2017/03/30 00:00 [received] PHST- 2017/12/05 00:00 [accepted] PHST- 2017/12/15 06:00 [entrez] PHST- 2017/12/15 06:00 [pubmed] PHST- 2018/07/19 06:00 [medline] AID - 27293 [pii] AID - 10.7554/eLife.27293 [doi] PST - epublish SO - Elife. 2017 Dec 14;6:e27293. doi: 10.7554/eLife.27293. PMID- 29171006 OWN - NLM STAT- MEDLINE DCOM- 20180914 LR - 20211001 IS - 1528-1167 (Electronic) IS - 0013-9580 (Print) IS - 0013-9580 (Linking) VI - 59 IP - 1 DP - 2018 Jan TI - BAD knockout provides metabolic seizure resistance in a genetic model of epilepsy with sudden unexplained death in epilepsy. PG - e1-e4 LID - 10.1111/epi.13960 [doi] AB - Metabolic alteration, either through the ketogenic diet (KD) or by genetic alteration of the BAD protein, can produce seizure protection in acute chemoconvulsant models of epilepsy. To assess the seizure-protective role of knocking out (KO) the Bad gene in a chronic epilepsy model, we used the Kcna1(-/-) model of epilepsy, which displays progressively increased seizure severity and recapitulates the early death seen in sudden unexplained death in epilepsy (SUDEP). Beginning on postnatal day 24 (P24), we continuously video monitored Kcna1(-/-) and Kcna1(-/-) Bad(-/-) double knockout mice to assess survival and seizure severity. We found that Kcna1(-/-) Bad(-/-) mice outlived Kcna1(-/-) mice by approximately 2 weeks. Kcna1(-/-) Bad(-/-) mice also spent significantly less time in seizure than Kcna1(-/-) mice on P24 and the day of death, showing that BadKO provides seizure resistance in a genetic model of chronic epilepsy. CI - Wiley Periodicals, Inc. © 2017 International League Against Epilepsy. FAU - Foley, Jeannine AU - Foley J AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Burnham, Veronica AU - Burnham V AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Tedoldi, Meghan AU - Tedoldi M AD - Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. FAU - Danial, Nika N AU - Danial NN AD - Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - R01 NS083844/NS/NINDS NIH HHS/United States GR - R01 NS102586/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20171123 PL - United States TA - Epilepsia JT - Epilepsia JID - 2983306R RN - 0 (Bad protein, mouse) RN - 0 (Kcna1 protein, mouse) RN - 0 (bcl-Associated Death Protein) RN - 147173-20-4 (Kv1.1 Potassium Channel) SB - IM MH - Age Factors MH - Animals MH - Brugada Syndrome/*etiology/*genetics/metabolism MH - Disease Models, Animal MH - Electroencephalography MH - *Epilepsy/complications/genetics/prevention & control MH - Female MH - Kv1.1 Potassium Channel/*genetics/metabolism MH - Male MH - Mice MH - Mice, Inbred C57BL MH - Mice, Knockout MH - bcl-Associated Death Protein/*deficiency/genetics PMC - PMC5760331 MID - NIHMS918025 OTO - NOTNLM OT - BAD OT - Kcna1 −/− OT - Kv1.1 OT - metabolic seizure resistance COIS- DISCLOSURE None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. EDAT- 2017/11/25 06:00 MHDA- 2018/09/15 06:00 CRDT- 2017/11/25 06:00 PHST- 2017/10/26 00:00 [accepted] PHST- 2017/11/25 06:00 [pubmed] PHST- 2018/09/15 06:00 [medline] PHST- 2017/11/25 06:00 [entrez] AID - 10.1111/epi.13960 [doi] PST - ppublish SO - Epilepsia. 2018 Jan;59(1):e1-e4. doi: 10.1111/epi.13960. Epub 2017 Nov 23. PMID- 29025729 OWN - NLM STAT- MEDLINE DCOM- 20190226 LR - 20191210 IS - 1522-1547 (Electronic) IS - 0193-1857 (Print) IS - 0193-1857 (Linking) VI - 314 IP - 1 DP - 2018 Jan 1 TI - Live cell imaging of cytosolic NADH/NAD(+) ratio in hepatocytes and liver slices. PG - G97-G108 LID - 10.1152/ajpgi.00093.2017 [doi] AB - Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD(+) ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD(+) ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD(+) ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD(+) ratio, and this increase is mitigated by the presence of NAD(+)-generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD(+) ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD(+) ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real-time measurements of the ratio in living, functioning liver cells, overcoming many limitations of previous methods for measuring this important redox parameter. The feasibility of using Peredox in liver slices is particularly attractive because slices allow preservation of hepatic microanatomy and metabolic zonation of hepatocytes. FAU - Masia, Ricard AU - Masia R AD - Department of Pathology and Laboratory Medicine, Massachusetts General Hospital , Boston, Massachusetts. AD - Department of Neurobiology, Harvard Medical School , Boston, Massachusetts. FAU - McCarty, William J AU - McCarty WJ AD - Department of Surgery, Massachusetts General Hospital , Boston, Massachusetts. FAU - Lahmann, Carolina AU - Lahmann C AD - Department of Neurobiology, Harvard Medical School , Boston, Massachusetts. FAU - Luther, Jay AU - Luther J AD - Gastrointestinal Unit, Massachusetts General Hospital , Boston, Massachusetts. FAU - Chung, Raymond T AU - Chung RT AD - Gastrointestinal Unit, Massachusetts General Hospital , Boston, Massachusetts. FAU - Yarmush, Martin L AU - Yarmush ML AD - Department of Surgery, Massachusetts General Hospital , Boston, Massachusetts. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School , Boston, Massachusetts. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - F32 NS093784/NS/NINDS NIH HHS/United States GR - T32 CA009216/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Validation Study DEP - 20171012 PL - United States TA - Am J Physiol Gastrointest Liver Physiol JT - American journal of physiology. Gastrointestinal and liver physiology JID - 100901227 RN - 0 (Biomarkers) RN - 0 (Luminescent Proteins) RN - 0U46U6E8UK (NAD) SB - IM MH - Animals MH - Biomarkers/metabolism MH - *Biosensing Techniques MH - Cytosol/*metabolism MH - *Energy Metabolism MH - Female MH - Genes, Reporter MH - Hep G2 Cells MH - Hepatocytes/*metabolism MH - Humans MH - In Vitro Techniques MH - Liver/*metabolism MH - Luminescent Proteins/biosynthesis/genetics MH - Male MH - Mice, Inbred C57BL MH - Microscopy, Fluorescence, Multiphoton/*methods MH - NAD/*metabolism MH - Oxidation-Reduction MH - Rats, Inbred Lew MH - Reproducibility of Results MH - Time Factors MH - Transfection PMC - PMC5866369 OTO - NOTNLM OT - NADH OT - alcohol OT - alcohol dehydrogenase OT - fluorescent biosensor OT - liver slice EDAT- 2017/10/14 06:00 MHDA- 2019/02/27 06:00 CRDT- 2017/10/14 06:00 PHST- 2017/10/14 06:00 [pubmed] PHST- 2019/02/27 06:00 [medline] PHST- 2017/10/14 06:00 [entrez] AID - ajpgi.00093.2017 [pii] AID - GI-00093-2017 [pii] AID - 10.1152/ajpgi.00093.2017 [doi] PST - ppublish SO - Am J Physiol Gastrointest Liver Physiol. 2018 Jan 1;314(1):G97-G108. doi: 10.1152/ajpgi.00093.2017. Epub 2017 Oct 12. PMID- 28768175 OWN - NLM STAT- MEDLINE DCOM- 20180417 LR - 20181215 IS - 1932-7420 (Electronic) IS - 1550-4131 (Print) IS - 1550-4131 (Linking) VI - 26 IP - 2 DP - 2017 Aug 1 TI - Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake. PG - 361-374.e4 LID - S1550-4131(17)30421-7 [pii] LID - 10.1016/j.cmet.2017.06.021 [doi] AB - Proper brain function requires a substantial energy supply, up to 20% of whole-body energy in humans, and brain activation produces large dynamic variations in energy demand. While local increases in cerebral blood flow are well known, the cellular responses to energy demand are controversial. During brain excitation, glycolysis of glucose to lactate temporarily exceeds the rate of mitochondrial fuel oxidation; although the increased energy demand occurs mainly within neurons, some have suggested this glycolysis occurs mainly in astrocytes, which then shuttle lactate to neurons as their primary fuel. Using metabolic biosensors in acute hippocampal slices and brains of awake mice, we find that neuronal metabolic responses to stimulation do not depend on astrocytic stimulation by glutamate release, nor do they require neuronal uptake of lactate; instead they reflect increased direct glucose consumption by neurons. Neuronal glycolysis temporarily outstrips oxidative metabolism, and provides a rapid response to increased energy demand. CI - Copyright © 2017 Elsevier Inc. All rights reserved. FAU - Díaz-García, Carlos Manlio AU - Díaz-García CM AD - Department of Neurobiology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. FAU - Mongeon, Rebecca AU - Mongeon R AD - Department of Neurobiology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. FAU - Lahmann, Carolina AU - Lahmann C AD - Department of Neurobiology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. FAU - Koveal, Dorothy AU - Koveal D AD - Department of Neurobiology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. FAU - Zucker, Hannah AU - Zucker H AD - Department of Neurobiology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. Electronic address: gary_yellen@hms.harvard.edu. LA - eng GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - F32 NS080455/NS/NINDS NIH HHS/United States GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - F32 NS093784/NS/NINDS NIH HHS/United States GR - F32 NS100331/NS/NINDS NIH HHS/United States PT - Journal Article PL - United States TA - Cell Metab JT - Cell metabolism JID - 101233170 RN - 33X04XA5AT (Lactic Acid) SB - IM MH - Animals MH - Glycolysis/*physiology MH - Hippocampus/cytology/*metabolism MH - Lactic Acid/*metabolism MH - Male MH - Mice MH - Neurons/cytology/*metabolism PMC - PMC5559896 MID - NIHMS890117 OTO - NOTNLM OT - astrocyte-neuron lactate shuttle OT - brain metabolism OT - glycolysis EDAT- 2017/08/03 06:00 MHDA- 2018/04/18 06:00 CRDT- 2017/08/03 06:00 PHST- 2016/11/04 00:00 [received] PHST- 2017/04/29 00:00 [revised] PHST- 2017/06/27 00:00 [accepted] PHST- 2017/08/03 06:00 [entrez] PHST- 2017/08/03 06:00 [pubmed] PHST- 2018/04/18 06:00 [medline] AID - S1550-4131(17)30421-7 [pii] AID - 10.1016/j.cmet.2017.06.021 [doi] PST - ppublish SO - Cell Metab. 2017 Aug 1;26(2):361-374.e4. doi: 10.1016/j.cmet.2017.06.021. PMID- 27177420 OWN - NLM STAT- MEDLINE DCOM- 20171106 LR - 20201209 IS - 2050-084X (Electronic) IS - 2050-084X (Linking) VI - 5 DP - 2016 May 13 TI - The leak channel NALCN controls tonic firing and glycolytic sensitivity of substantia nigra pars reticulata neurons. LID - e15271 [pii] LID - 10.7554/eLife.15271 [doi] AB - Certain neuron types fire spontaneously at high rates, an ability that is crucial for their function in brain circuits. The spontaneously active GABAergic neurons of the substantia nigra pars reticulata (SNr), a major output of the basal ganglia, provide tonic inhibition of downstream brain areas. A depolarizing 'leak' current supports this firing pattern, but its molecular basis remains poorly understood. To understand how SNr neurons maintain tonic activity, we used single-cell RNA sequencing to determine the transcriptome of individual mouse SNr neurons. We discovered that SNr neurons express the sodium leak channel, NALCN, and that SNr neurons lacking NALCN have impaired spontaneous firing. In addition, NALCN is involved in the modulation of excitability by changes in glycolysis and by activation of muscarinic acetylcholine receptors. Our findings suggest that disruption of NALCN could impair the basal ganglia circuit, which may underlie the severe motor deficits in humans carrying mutations in NALCN. FAU - Lutas, Andrew AU - Lutas A AUID- ORCID: 0000-0002-6991-2898 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Lahmann, Carolina AU - Lahmann C AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Soumillon, Magali AU - Soumillon M AD - Broad Institute, Cambridge, United States. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, United States. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - F31 NS077633/NS/NINDS NIH HHS/United States GR - P30 NS072030/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States PT - Journal Article DEP - 20160513 PL - England TA - Elife JT - eLife JID - 101579614 RN - 0 (Ion Channels) RN - 0 (Membrane Proteins) RN - 0 (NALCN protein, mouse) RN - 0 (Nerve Tissue Proteins) SB - IM MH - *Action Potentials MH - Animals MH - GABAergic Neurons/metabolism/*physiology MH - Gene Expression Profiling MH - *Glycolysis MH - Ion Channels/*metabolism MH - Membrane Proteins MH - Mice MH - Nerve Tissue Proteins/*metabolism MH - Pars Reticulata/*physiology MH - Sequence Analysis, RNA MH - Single-Cell Analysis PMC - PMC4902561 OTO - NOTNLM OT - basal ganglia OT - leak current OT - metabolism and excitability OT - mouse OT - neuroscience OT - spontaneous firing COIS- The authors declare that no competing interests exist. EDAT- 2016/05/14 06:00 MHDA- 2017/11/07 06:00 CRDT- 2016/05/14 06:00 PHST- 2016/02/15 00:00 [received] PHST- 2016/05/12 00:00 [accepted] PHST- 2016/05/14 06:00 [entrez] PHST- 2016/05/14 06:00 [pubmed] PHST- 2017/11/07 06:00 [medline] AID - e15271 [pii] AID - 15271 [pii] AID - 10.7554/eLife.15271 [doi] PST - epublish SO - Elife. 2016 May 13;5:e15271. doi: 10.7554/eLife.15271. PMID- 26941646 OWN - NLM STAT- PubMed-not-MEDLINE DCOM- 20160305 LR - 20200929 IS - 1663-9812 (Print) IS - 1663-9812 (Electronic) IS - 1663-9812 (Linking) VI - 7 DP - 2016 TI - Corrigendum: A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging. PG - 46 LID - 10.3389/fphar.2016.00046 [doi] LID - 46 AB - [This corrects the article on p. 56 in vol. 5, PMID: 24765076.]. FAU - Chen, Yao AU - Chen Y AD - Howard Hughes Medical InstituteBoston, MA, USA; Department of Neurobiology, Harvard Medical SchoolBoston, MA, USA. FAU - Saulnier, Jessica L AU - Saulnier JL AD - Howard Hughes Medical InstituteBoston, MA, USA; Department of Neurobiology, Harvard Medical SchoolBoston, MA, USA. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School Boston, MA, USA. FAU - Sabatini, Bernardo L AU - Sabatini BL AD - Howard Hughes Medical InstituteBoston, MA, USA; Department of Neurobiology, Harvard Medical SchoolBoston, MA, USA. LA - eng GR - R01 NS046579/NS/NINDS NIH HHS/United States PT - Published Erratum DEP - 20160225 PL - Switzerland TA - Front Pharmacol JT - Frontiers in pharmacology JID - 101548923 EFR - Front Pharmacol. 2014;5:56. PMID: 24765076 PMC - PMC4766420 OTO - NOTNLM OT - FLIM OT - FLIM-AKAR OT - GPCR OT - PKA OT - cAMP OT - dendritic spine OT - glutamate OT - neuromodulation EDAT- 2016/03/05 06:00 MHDA- 2016/03/05 06:01 CRDT- 2016/03/05 06:00 PHST- 2016/02/17 00:00 [received] PHST- 2016/02/18 00:00 [accepted] PHST- 2016/03/05 06:00 [entrez] PHST- 2016/03/05 06:00 [pubmed] PHST- 2016/03/05 06:01 [medline] AID - 10.3389/fphar.2016.00046 [doi] PST - epublish SO - Front Pharmacol. 2016 Feb 25;7:46. doi: 10.3389/fphar.2016.00046. eCollection 2016. PMID- 26857245 OWN - NLM STAT- MEDLINE DCOM- 20170710 LR - 20181215 IS - 1557-7716 (Electronic) IS - 1523-0864 (Print) IS - 1523-0864 (Linking) VI - 25 IP - 10 DP - 2016 Oct 1 TI - Cytosolic NADH-NAD(+) Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging. PG - 553-63 LID - 10.1089/ars.2015.6593 [doi] AB - AIM: Cytosolic NADH-NAD(+) redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD(+) redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. RESULTS: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD(+) ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. INNOVATION: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. CONCLUSION: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD(+) ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553-563. FAU - Mongeon, Rebecca AU - Mongeon R AD - Department of Neurobiology, Harvard Medical School , Boston, Massachusetts. FAU - Venkatachalam, Veena AU - Venkatachalam V AD - Department of Neurobiology, Harvard Medical School , Boston, Massachusetts. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School , Boston, Massachusetts. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - F32 NS080455/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - T32 GM007753/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20160318 PL - United States TA - Antioxid Redox Signal JT - Antioxidants & redox signaling JID - 100888899 RN - 0U46U6E8UK (NAD) RN - 33X04XA5AT (Lactic Acid) RN - IY9XDZ35W2 (Glucose) SB - IM MH - Animals MH - Astrocytes/metabolism/ultrastructure MH - *Biosensing Techniques MH - Cytosol/*metabolism/ultrastructure MH - Glucose/metabolism MH - Glycolysis MH - Hippocampus/diagnostic imaging/*metabolism/ultrastructure MH - Lactic Acid/metabolism MH - Mice MH - NAD/isolation & purification/*metabolism MH - Neurons/metabolism/ultrastructure MH - Oxidation-Reduction PMC - PMC5041510 COIS- Author Disclosure Statement G.Y. has licensed the digitized FLIM technology to Thorlabs, Inc., for whom he is also a consultant. All other authors state that no competing financial interests exist. EDAT- 2016/02/10 06:00 MHDA- 2017/07/14 06:00 CRDT- 2016/02/10 06:00 PHST- 2016/02/10 06:00 [entrez] PHST- 2016/02/10 06:00 [pubmed] PHST- 2017/07/14 06:00 [medline] AID - 10.1089/ars.2015.6593 [pii] AID - 10.1089/ars.2015.6593 [doi] PST - ppublish SO - Antioxid Redox Signal. 2016 Oct 1;25(10):553-63. doi: 10.1089/ars.2015.6593. Epub 2016 Mar 18. PMID- 26590798 OWN - NLM STAT- MEDLINE DCOM- 20160919 LR - 20181113 IS - 1872-6844 (Electronic) IS - 0920-1211 (Print) IS - 0920-1211 (Linking) VI - 118 DP - 2015 Dec TI - Variants in KCNJ11 and BAD do not predict response to ketogenic dietary therapies for epilepsy. PG - 22-8 LID - S0920-1211(15)30058-9 [pii] LID - 10.1016/j.eplepsyres.2015.10.003 [doi] AB - In the absence of specific metabolic disorders, predictors of response to ketogenic dietary therapies (KDT) are unknown. We aimed to determine whether variants in established candidate genes KCNJ11 and BAD influence response to KDT. We sequenced KCNJ11 and BAD in individuals without previously-known glucose transporter type 1 deficiency syndrome or other metabolic disorders, who received KDT for epilepsy. Hospital records were used to obtain demographic and clinical data. Two response phenotypes were used: ≥ 50% seizure reduction and seizure-freedom at 3-month follow-up. Case/control association tests were conducted with KCNJ11 and BAD variants with minor allele frequency (MAF)>0.01, using PLINK. Response to KDT in individuals with variants with MAF<0.01 was evaluated. 303 Individuals had KCNJ11 and 246 individuals had BAD sequencing data and diet response data. Six SNPs in KCNJ11 and two in BAD had MAF>0.01. Eight variants in KCNJ11 and seven in BAD (of which three were previously-unreported) had MAF<0.01. No significant results were obtained from association analyses, with either KDT response phenotype. P-values were similar when accounting for ethnicity using a stratified Cochran-Mantel-Haenszel test. There did not seem to be a consistent effect of rare variants on response to KDT, although the cohort size was too small to assess significance. Common variants in KCNJ11 and BAD do not predict response to KDT for epilepsy. We can exclude, with 80% power, association from variants with a MAF of >0.05 and effect size >3. A larger sample size is needed to detect associations from rare variants or those with smaller effect sizes. CI - Copyright © 2015 Elsevier B.V. All rights reserved. FAU - Schoeler, Natasha E AU - Schoeler NE AD - NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom; UCL Institute of Child Health, London, United Kingdom. Electronic address: n.schoeler.10@ucl.ac.uk. FAU - Leu, Costin AU - Leu C AD - NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom. FAU - White, Jon AU - White J AD - Department of Genetics Environment and Evolution, UCL Genetics Institute, London, United Kingdom. FAU - Plagnol, Vincent AU - Plagnol V AD - Department of Statistical Genetics, University College London, London, United Kingdom. FAU - Ellard, Sian AU - Ellard S AD - Molecular Genetics, University of Exeter Medical School, Exeter, United Kingdom. FAU - Matarin, Mar AU - Matarin M AD - NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Thiele, Elizabeth A AU - Thiele EA AD - Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. FAU - Mackay, Mark AU - Mackay M AD - Royal Children's Hospital, Melbourne, Australia; Murdoch Children's Research Institute, Melbourne, Australia. FAU - McMahon, Jacinta M AU - McMahon JM AD - Epilepsy Research Centre, The University of Melbourne, Austin Health, Melbourne, Australia. FAU - Scheffer, Ingrid E AU - Scheffer IE AD - Royal Children's Hospital, Melbourne, Australia; Departments of Medicine and Paediatrics, The University of Melbourne, Melbourne, Australia; Florey Institute of Neurosciences and Mental Health, Austin Health, Melbourne, Australia. FAU - Sander, Josemir W AU - Sander JW AD - NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom; Epilepsy Society, Chalfont St Peter, United Kingdom; Stichting Epilepsie Instellingen Nederland (SEIN), Heemstede, The Netherlands. FAU - Cross, J Helen AU - Cross JH AD - UCL Institute of Child Health, London, United Kingdom; Great Ormond Street Hospital for Children, London, United Kingdom; Young Epilepsy, Lingfield, United Kingdom. FAU - Sisodiya, Sanjay M AU - Sisodiya SM AD - NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom; Epilepsy Society, Chalfont St Peter, United Kingdom. LA - eng GR - 098395/Wellcome Trust/United Kingdom GR - 084730/Wellcome Trust/United Kingdom PT - Journal Article PT - Multicenter Study PT - Research Support, Non-U.S. Gov't DEP - 20151024 PL - Netherlands TA - Epilepsy Res JT - Epilepsy research JID - 8703089 RN - 0 (BAD protein, human) RN - 0 (Kir6.2 channel) RN - 0 (Potassium Channels, Inwardly Rectifying) RN - 0 (bcl-Associated Death Protein) SB - IM MH - Analysis of Variance MH - Child MH - Child, Preschool MH - Cohort Studies MH - Diet, Ketogenic/*methods MH - Electroencephalography MH - Epilepsy/*diet therapy/*genetics MH - Female MH - Genetic Association Studies MH - Genetic Testing MH - Genotype MH - Humans MH - Male MH - Pharmacogenetics MH - Polymorphism, Single Nucleotide/*genetics MH - Potassium Channels, Inwardly Rectifying/*genetics MH - United Kingdom MH - bcl-Associated Death Protein/*genetics PMC - PMC4819482 OTO - NOTNLM OT - BAD OT - Epilepsy OT - Genetic biomarker OT - KCNJ11 OT - Ketogenic diet OT - Seizures EDAT- 2015/11/23 06:00 MHDA- 2016/09/20 06:00 CRDT- 2015/11/23 06:00 PHST- 2015/06/06 00:00 [received] PHST- 2015/08/24 00:00 [revised] PHST- 2015/10/20 00:00 [accepted] PHST- 2015/11/23 06:00 [entrez] PHST- 2015/11/23 06:00 [pubmed] PHST- 2016/09/20 06:00 [medline] AID - S0920-1211(15)30058-9 [pii] AID - 10.1016/j.eplepsyres.2015.10.003 [doi] PST - ppublish SO - Epilepsy Res. 2015 Dec;118:22-8. doi: 10.1016/j.eplepsyres.2015.10.003. Epub 2015 Oct 24. PMID- 26079046 OWN - NLM STAT- MEDLINE DCOM- 20160523 LR - 20181113 IS - 1879-0402 (Electronic) IS - 1367-5931 (Print) IS - 1367-5931 (Linking) VI - 27 DP - 2015 Aug TI - Quantitative two-photon imaging of fluorescent biosensors. PG - 24-30 LID - S1367-5931(15)00062-9 [pii] LID - 10.1016/j.cbpa.2015.05.024 [doi] AB - Fluorescent biosensors are now routinely imaged using two-photon microscopy in intact tissue, for instance, in brain slices and brains in living animals. But most studies measure temporal variation-for example, calcium transients in response to neuronal activity-rather than calibrated levels of biosensor occupancy (and thus levels of the sensed analyte). True quantitative measurements are challenging, since it is difficult or impossible to calibrate a sensor's dose-response in situ, and difficult to compare the optical signals from tissue to those during in vitro calibration. Ratiometric measurements (at two wavelengths) are complicated by variations in laser power and by wavelength-dependent attenuation in tissue. For some biosensors, fluorescence lifetime imaging microscopy (FLIM) provides a valuable alternative that gives well-calibrated measurements of analyte levels. CI - Copyright © 2015 Elsevier Ltd. All rights reserved. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, United States. Electronic address: gary_yellen@hms.harvard.edu. FAU - Mongeon, Rebecca AU - Mongeon R AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, United States. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - F32 NS080455/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - DP1 EB016985/DP/NCCDPHP CDC HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Review DEP - 20150612 PL - England TA - Curr Opin Chem Biol JT - Current opinion in chemical biology JID - 9811312 RN - 0 (Fluorescent Dyes) RN - 0 (Luminescent Proteins) SB - IM MH - Animals MH - Biosensing Techniques/instrumentation/*methods MH - Brain/metabolism/pathology MH - Calibration MH - Cell Line MH - Cell Tracking/methods MH - Fluorescence Resonance Energy Transfer/*methods MH - Fluorescent Dyes/*chemistry MH - Humans MH - Luminescent Proteins/*chemistry/genetics MH - Microscopy, Fluorescence, Multiphoton/*methods MH - Molecular Imaging/instrumentation/*methods MH - Protein Binding PMC - PMC4553104 MID - NIHMS696811 EDAT- 2015/06/17 06:00 MHDA- 2016/05/24 06:00 CRDT- 2015/06/17 06:00 PHST- 2015/02/27 00:00 [received] PHST- 2015/05/11 00:00 [revised] PHST- 2015/05/21 00:00 [accepted] PHST- 2015/06/17 06:00 [entrez] PHST- 2015/06/17 06:00 [pubmed] PHST- 2016/05/24 06:00 [medline] AID - S1367-5931(15)00062-9 [pii] AID - 10.1016/j.cbpa.2015.05.024 [doi] PST - ppublish SO - Curr Opin Chem Biol. 2015 Aug;27:24-30. doi: 10.1016/j.cbpa.2015.05.024. Epub 2015 Jun 12. PMID- 25472961 OWN - NLM STAT- MEDLINE DCOM- 20150320 LR - 20200930 IS - 1522-1563 (Electronic) IS - 0363-6143 (Print) IS - 0363-6143 (Linking) VI - 308 IP - 3 DP - 2015 Feb 1 TI - The inward rectifier potassium channel Kir2.1 is expressed in mouse neutrophils from bone marrow and liver. PG - C264-76 LID - 10.1152/ajpcell.00176.2014 [doi] AB - Neutrophils are phagocytic cells that play a critical role in innate immunity by destroying bacterial pathogens. Channels belonging to the inward rectifier potassium channel subfamily 2 (Kir2 channels) have been described in other phagocytes (monocytes/macrophages and eosinophils) and in hematopoietic precursors of phagocytes. Their physiological function in these cells remains unclear, but some evidence suggests a role in growth factor-dependent proliferation and development. Expression of functional Kir2 channels has not been definitively demonstrated in mammalian neutrophils. Here, we show by RT-PCR that neutrophils from mouse bone marrow and liver express mRNA for the Kir2 subunit Kir2.1 but not for other subunits (Kir2.2, Kir2.3, and Kir2.4). In electrophysiological experiments, resting (unstimulated) neutrophils from mouse bone marrow and liver exhibit a constitutively active, external K(+)-dependent, strong inwardly rectifying current that constitutes the dominant current. The reversal potential is dependent on the external K(+) concentration in a Nernstian fashion, as expected for a K(+)-selective current. The current is not altered by changes in external or internal pH, and it is blocked by Ba(2+), Cs(+), and the Kir2-selective inhibitor ML133. The single-channel conductance is in agreement with previously reported values for Kir2.1 channels. These properties are characteristic of homomeric Kir2.1 channels. Current density in short-term cultures of bone marrow neutrophils is decreased in the absence of growth factors that are important for neutrophil proliferation [granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF)]. These results demonstrate that mouse neutrophils express functional Kir2.1 channels and suggest that these channels may be important for neutrophil function, possibly in a growth factor-dependent manner. CI - Copyright © 2015 the American Physiological Society. FAU - Masia, Ricard AU - Masia R AD - Department of Pathology and Laboratory Medicine, Massachusetts General Hospital, Boston, Massachusetts; and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts rmasia@mgh.harvard.edu. FAU - Krause, Daniela S AU - Krause DS AD - Department of Pathology and Laboratory Medicine, Massachusetts General Hospital, Boston, Massachusetts; and. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. LA - eng GR - T32 CA009216/CA/NCI NIH HHS/United States GR - K08 CA138916-02/CA/NCI NIH HHS/United States GR - T32 CA09216/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20141203 PL - United States TA - Am J Physiol Cell Physiol JT - American journal of physiology. Cell physiology JID - 100901225 RN - 0 (Kir2.1 channel) RN - 0 (Potassium Channels, Inwardly Rectifying) SB - IM MH - Animals MH - Bone Marrow/*metabolism MH - Cells, Cultured MH - Female MH - Gene Expression Regulation MH - Liver/cytology/*metabolism MH - Male MH - Mice MH - Mice, Inbred C57BL MH - Neutrophils/*metabolism MH - Potassium Channels, Inwardly Rectifying/*biosynthesis PMC - PMC4312842 OTO - NOTNLM OT - Kir2 channels OT - granulocyte colony-stimulating factor OT - inward rectifier potassium channel OT - neutrophil EDAT- 2014/12/05 06:00 MHDA- 2015/03/21 06:00 CRDT- 2014/12/05 06:00 PHST- 2014/12/05 06:00 [entrez] PHST- 2014/12/05 06:00 [pubmed] PHST- 2015/03/21 06:00 [medline] AID - ajpcell.00176.2014 [pii] AID - C-00176-2014 [pii] AID - 10.1152/ajpcell.00176.2014 [doi] PST - ppublish SO - Am J Physiol Cell Physiol. 2015 Feb 1;308(3):C264-76. doi: 10.1152/ajpcell.00176.2014. Epub 2014 Dec 3. PMID- 25471572 OWN - NLM STAT- MEDLINE DCOM- 20150219 LR - 20191220 IS - 1529-2401 (Electronic) IS - 0270-6474 (Print) IS - 0270-6474 (Linking) VI - 34 IP - 49 DP - 2014 Dec 3 TI - Metabolism regulates the spontaneous firing of substantia nigra pars reticulata neurons via KATP and nonselective cation channels. PG - 16336-47 LID - 10.1523/JNEUROSCI.1357-14.2014 [doi] AB - Neurons use glucose to fuel glycolysis and provide substrates for mitochondrial respiration, but neurons can also use alternative fuels that bypass glycolysis and feed directly into mitochondria. To determine whether neuronal pacemaking depends on active glucose metabolism, we switched the metabolic fuel from glucose to alternative fuels, lactate or β-hydroxybutyrate, while monitoring the spontaneous firing of GABAergic neurons in mouse substantia nigra pars reticulata (SNr) brain slices. We found that alternative fuels, in the absence of glucose, sustained SNr spontaneous firing at basal rates, but glycolysis may still be supported by glycogen in the absence of glucose. To prevent any glycogen-fueled glycolysis, we directly inhibited glycolysis using either 2-deoxyglucose or iodoacetic acid. Inhibiting glycolysis in the presence of alternative fuels lowered SNr firing to a slower sustained firing rate. Surprisingly, we found that the decrease in SNr firing was not mediated by ATP-sensitive potassium (KATP) channel activity, but if we lowered the perfusion flow rate or omitted the alternative fuel, KATP channels were activated and could silence SNr firing. The KATP-independent slowing of SNr firing that occurred with glycolytic inhibition in the presence of alternative fuels was consistent with a decrease in a nonselective cationic conductance. Although mitochondrial metabolism alone can prevent severe energy deprivation and KATP channel activation in SNr neurons, active glucose metabolism appears important for keeping open a class of ion channels that is crucial for the high spontaneous firing rate of SNr neurons. CI - Copyright © 2014 the authors 0270-6474/14/3416336-12$15.00/0. FAU - Lutas, Andrew AU - Lutas A AUID- ORCID: 0000-0002-6991-2898 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, and. FAU - Birnbaumer, Lutz AU - Birnbaumer L AUID- ORCID: 0000-0002-0775-8661 AD - Laboratory of Neurobiology, National Institute of Environmental Research, National Institutes of Health, Research Triangle Park, North Carolina 27709. FAU - Yellen, Gary AU - Yellen G AUID- ORCID: 0000-0003-4228-7866 AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, and gary_yellen@hms.harvard.edu. LA - eng GR - F31 NS077633/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - Z01 ES101684/Intramural NIH HHS/United States GR - Z01-ES-101684/ES/NIEHS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, N.I.H., Intramural PL - United States TA - J Neurosci JT - The Journal of neuroscience : the official journal of the Society for Neuroscience JID - 8102140 RN - 0 (KATP Channels) RN - 0 (Transient Receptor Potential Channels) RN - 33X04XA5AT (Lactic Acid) RN - 9G2MP84A8W (Deoxyglucose) RN - TZP1275679 (3-Hydroxybutyric Acid) RN - WF5188V710 (Iodoacetic Acid) SB - IM MH - 3-Hydroxybutyric Acid/metabolism MH - Action Potentials/*physiology MH - Animals MH - Deoxyglucose/pharmacology MH - Female MH - GABAergic Neurons/*physiology MH - Glycolysis/drug effects/physiology MH - Iodoacetic Acid/pharmacology MH - KATP Channels/*physiology MH - Lactic Acid/metabolism MH - Male MH - Mice MH - Neurons/*metabolism MH - Pars Reticulata/*cytology/*physiology MH - Transient Receptor Potential Channels/*physiology PMC - PMC4252546 OTO - NOTNLM OT - KATP OT - Trp channel OT - excitability OT - glycolysis EDAT- 2014/12/05 06:00 MHDA- 2015/02/20 06:00 CRDT- 2014/12/05 06:00 PHST- 2014/12/05 06:00 [entrez] PHST- 2014/12/05 06:00 [pubmed] PHST- 2015/02/20 06:00 [medline] AID - 34/49/16336 [pii] AID - 1357-14 [pii] AID - 10.1523/JNEUROSCI.1357-14.2014 [doi] PST - ppublish SO - J Neurosci. 2014 Dec 3;34(49):16336-47. doi: 10.1523/JNEUROSCI.1357-14.2014. PMID- 25416365 OWN - NLM STAT- MEDLINE DCOM- 20150804 LR - 20181113 IS - 1557-7988 (Electronic) IS - 0076-6879 (Print) IS - 0076-6879 (Linking) VI - 547 DP - 2014 TI - Imaging changes in the cytosolic ATP-to-ADP ratio. PG - 355-71 LID - B978-0-12-801415-8.00017-5 [pii] LID - 10.1016/B978-0-12-801415-8.00017-5 [doi] AB - Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside the cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We, therefore, developed a genetically encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here, we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy. FAU - Tantama, Mathew AU - Tantama M AD - Department of Chemistry, Purdue University, West Lafayette, Indiana, USA. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. Electronic address: gary_yellen@hms.harvard.edu. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - F32 NS066613/NS/NINDS NIH HHS/United States PT - Journal Article PL - United States TA - Methods Enzymol JT - Methods in enzymology JID - 0212271 RN - 61D2G4IYVH (Adenosine Diphosphate) RN - 8L70Q75FXE (Adenosine Triphosphate) SB - IM MH - Adenosine Diphosphate/*analysis/metabolism MH - Adenosine Triphosphate/*analysis/metabolism MH - Animals MH - *Biosensing Techniques MH - Brain/cytology/metabolism MH - Calibration MH - Cells, Cultured MH - Cytosol/*metabolism MH - Energy Metabolism MH - HEK293 Cells MH - Humans MH - Image Processing, Computer-Assisted MH - Mice MH - Microscopy, Fluorescence/instrumentation/*methods PMC - PMC4323350 MID - NIHMS660422 OTO - NOTNLM OT - ADP OT - ATP OT - Energy OT - Fluorescence OT - Genetically encoded OT - Imaging OT - Metabolism OT - Microscopy OT - Ratiometric OT - Sensor EDAT- 2014/11/25 06:00 MHDA- 2015/08/05 06:00 CRDT- 2014/11/23 06:00 PHST- 2014/11/23 06:00 [entrez] PHST- 2014/11/25 06:00 [pubmed] PHST- 2015/08/05 06:00 [medline] AID - B978-0-12-801415-8.00017-5 [pii] AID - 10.1016/B978-0-12-801415-8.00017-5 [doi] PST - ppublish SO - Methods Enzymol. 2014;547:355-71. doi: 10.1016/B978-0-12-801415-8.00017-5. PMID- 25009227 OWN - NLM STAT- MEDLINE DCOM- 20150728 LR - 20211021 IS - 2050-084X (Electronic) IS - 2050-084X (Linking) VI - 3 DP - 2014 Jul 9 TI - Quantitative determinants of aerobic glycolysis identify flux through the enzyme GAPDH as a limiting step. LID - 10.7554/eLife.03342 [doi] LID - e03342 AB - Aerobic glycolysis or the Warburg Effect (WE) is characterized by the increased metabolism of glucose to lactate. It remains unknown what quantitative changes to the activity of metabolism are necessary and sufficient for this phenotype. We developed a computational model of glycolysis and an integrated analysis using metabolic control analysis (MCA), metabolomics data, and statistical simulations. We identified and confirmed a novel mode of regulation specific to aerobic glycolysis where flux through GAPDH, the enzyme separating lower and upper glycolysis, is the rate-limiting step in the pathway and the levels of fructose (1,6) bisphosphate (FBP), are predictive of the rate and control points in glycolysis. Strikingly, negative flux control was found and confirmed for several steps thought to be rate-limiting in glycolysis. Together, these findings enumerate the biochemical determinants of the WE and suggest strategies for identifying the contexts in which agents that target glycolysis might be most effective. FAU - Shestov, Alexander A AU - Shestov AA AD - Division of Nutritional Sciences, Cornell University, Ithaca, United States. FAU - Liu, Xiaojing AU - Liu X AD - Division of Nutritional Sciences, Cornell University, Ithaca, United States. FAU - Ser, Zheng AU - Ser Z AD - Division of Nutritional Sciences, Cornell University, Ithaca, United States. FAU - Cluntun, Ahmad A AU - Cluntun AA AD - Field of Biochemistry and Molecular Cell Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States. FAU - Hung, Yin P AU - Hung YP AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Huang, Lei AU - Huang L AD - Field of Computational Biology, Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, United States. FAU - Kim, Dongsung AU - Kim D AD - Field of Biochemistry and Molecular Cell Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States. FAU - Le, Anne AU - Le A AD - Department of Pathology and Oncology, Johns Hopkins University School of Medicine, Baltimore, United States. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, United States. FAU - Albeck, John G AU - Albeck JG AD - Department of Cell Biology, Harvard Medical School, Boston, United States. FAU - Locasale, Jason W AU - Locasale JW AUID- ORCID: 0000-0002-7766-3502 AD - Division of Nutritional Sciences, Cornell University, Ithaca, United States. LA - eng GR - R01AI110613/AI/NIAID NIH HHS/United States GR - R00CA168997/CA/NCI NIH HHS/United States GR - P30 DK079637/DK/NIDDK NIH HHS/United States GR - R00 CA168997/CA/NCI NIH HHS/United States GR - DP1 EB016985/EB/NIBIB NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20140709 PL - England TA - Elife JT - eLife JID - 101579614 RN - 0 (Fructosediphosphates) RN - 0 (Glucose Transport Proteins, Facilitative) RN - 33X04XA5AT (Lactic Acid) RN - EC 1.1.1.27 (L-Lactate Dehydrogenase) RN - EC 1.2.1.12 (Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)) RN - IY9XDZ35W2 (Glucose) RN - M7522JYX1H (fructose-1,6-diphosphate) SB - IM MH - Computational Biology/methods MH - Fructosediphosphates/chemistry MH - Glucose/chemistry MH - Glucose Transport Proteins, Facilitative/metabolism MH - Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/*metabolism MH - Glycolysis MH - HCT116 Cells MH - Humans MH - L-Lactate Dehydrogenase/metabolism MH - Lactic Acid/chemistry MH - Mass Spectrometry MH - Metabolomics/methods MH - Models, Chemical MH - Monte Carlo Method MH - Phenotype MH - Phosphorylation PMC - PMC4118620 OTO - NOTNLM OT - biochemistry OT - glucose OT - glycolysis OT - human OT - human biology OT - mass spectrometry OT - mathematical modeling OT - medicine OT - metabolism OT - metabolomics COIS- JWL: A patent related to this work has been filed. US Provisional Patent Appln. No. 61/908,953. The other authors declare that no competing interests exist. EDAT- 2014/07/11 06:00 MHDA- 2015/07/29 06:00 CRDT- 2014/07/11 06:00 PHST- 2014/05/11 00:00 [received] PHST- 2014/07/08 00:00 [accepted] PHST- 2014/07/11 06:00 [entrez] PHST- 2014/07/11 06:00 [pubmed] PHST- 2015/07/29 06:00 [medline] AID - 03342 [pii] AID - 10.7554/eLife.03342 [doi] PST - epublish SO - Elife. 2014 Jul 9;3:e03342. doi: 10.7554/eLife.03342. PMID- 24765076 OWN - NLM STAT- PubMed-not-MEDLINE DCOM- 20140425 LR - 20211021 IS - 1663-9812 (Print) IS - 1663-9812 (Electronic) IS - 1663-9812 (Linking) VI - 5 DP - 2014 TI - A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging. PG - 56 LID - 10.3389/fphar.2014.00056 [doi] LID - 56 AB - Neuromodulators have profound effects on behavior, but the dynamics of their intracellular effectors has remained unclear. Most neuromodulators exert their function via G-protein-coupled receptors (GPCRs). One major challenge for understanding neuromodulator action is the lack of dynamic readouts of the biochemical signals produced by GPCR activation. The adenylate cyclase/cyclic AMP/protein kinase A (PKA) module is a central component of such biochemical signaling. This module is regulated by several behaviorally important neuromodulator receptors. Furthermore, PKA activity is necessary for the induction of many forms of synaptic plasticity as well as for the formation of long-term memory. In order to monitor PKA activity in brain tissue, we have developed a 2-photon fluorescence lifetime imaging microscopy (2pFLIM) compatible PKA sensor termed FLIM-AKAR, which is based on the ratiometric FRET sensor AKAR3. FLIM-AKAR shows a large dynamic range and little pH sensitivity. In addition, it is a rapidly diffusible cytoplasmic protein that specifically reports net PKA activity in situ. FLIM-AKAR expresses robustly in various brain regions with multiple transfection methods, can be targeted to genetically identified cell types, and responds to activation of both endogenous GPCRs and spatial-temporally specific delivery of glutamate. Initial experiments reveal differential regulation of PKA activity across subcellular compartments in response to neuromodulator inputs. Therefore, the reporter FLIM-AKAR, coupled with 2pFLIM, enables the study of PKA activity in response to neuromodulator inputs in genetically identified neurons in the brain, and sheds light on the intracellular dynamics of endogenous GPCR activation. FAU - Chen, Yao AU - Chen Y AD - Howard Hughes Medical Institute Boston, MA, USA ; Department of Neurobiology, Harvard Medical School Boston, MA, USA. FAU - Saulnier, Jessica L AU - Saulnier JL AD - Howard Hughes Medical Institute Boston, MA, USA ; Department of Neurobiology, Harvard Medical School Boston, MA, USA. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School Boston, MA, USA. FAU - Sabatini, Bernardo L AU - Sabatini BL AD - Howard Hughes Medical Institute Boston, MA, USA ; Department of Neurobiology, Harvard Medical School Boston, MA, USA. LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - R01 NS046579/NS/NINDS NIH HHS/United States GR - T32 NS007484/NS/NINDS NIH HHS/United States PT - Journal Article DEP - 20140402 PL - Switzerland TA - Front Pharmacol JT - Frontiers in pharmacology JID - 101548923 EIN - Front Pharmacol. 2016;7:46. PMID: 26941646 PMC - PMC3980114 OTO - NOTNLM OT - FLIM OT - FLIM-AKAR OT - GPCR OT - PKA OT - cAMP OT - dendritic spine OT - glutamate OT - neuromodulation EDAT- 2014/04/26 06:00 MHDA- 2014/04/26 06:01 CRDT- 2014/04/26 06:00 PHST- 2014/01/30 00:00 [received] PHST- 2014/03/14 00:00 [accepted] PHST- 2014/04/26 06:00 [entrez] PHST- 2014/04/26 06:00 [pubmed] PHST- 2014/04/26 06:01 [medline] AID - 10.3389/fphar.2014.00056 [doi] PST - epublish SO - Front Pharmacol. 2014 Apr 2;5:56. doi: 10.3389/fphar.2014.00056. eCollection 2014. PMID- 24096541 OWN - NLM STAT- MEDLINE DCOM- 20140501 LR - 20220309 IS - 2041-1723 (Electronic) IS - 2041-1723 (Linking) VI - 4 DP - 2013 TI - Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio. PG - 2550 LID - 10.1038/ncomms3550 [doi] AB - The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research. FAU - Tantama, Mathew AU - Tantama M AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115, USA. FAU - Martínez-François, Juan Ramón AU - Martínez-François JR FAU - Mongeon, Rebecca AU - Mongeon R FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - EB016985/EB/NIBIB NIH HHS/United States GR - F32NS080455/NS/NINDS NIH HHS/United States GR - F32 NS066613/NS/NINDS NIH HHS/United States GR - F32 NS080455/NS/NINDS NIH HHS/United States GR - DP1 EB016985/EB/NIBIB NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PL - England TA - Nat Commun JT - Nature communications JID - 101528555 RN - 0 (Archaeal Proteins) RN - 0 (GlnK1 protein, Methanosarcina mazei) RN - 0 (KATP Channels) RN - 0 (Recombinant Fusion Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 61D2G4IYVH (Adenosine Diphosphate) RN - 8L70Q75FXE (Adenosine Triphosphate) SB - IM MH - Adenosine Diphosphate/*analysis/biosynthesis MH - Adenosine Triphosphate/*analysis/biosynthesis MH - Animals MH - Archaeal Proteins/genetics/metabolism MH - Astrocytes/*metabolism/ultrastructure MH - *Biosensing Techniques MH - Cell Line MH - Embryo, Mammalian MH - Energy Metabolism/physiology MH - Escherichia coli/genetics/metabolism MH - Green Fluorescent Proteins/analysis/genetics/metabolism MH - Humans MH - KATP Channels/genetics/metabolism MH - Mice MH - Microscopy, Fluorescence, Multiphoton MH - Molecular Imaging MH - Neurons/*metabolism/ultrastructure MH - Primary Cell Culture MH - Recombinant Fusion Proteins/genetics/metabolism MH - Single-Cell Analysis PMC - PMC3852917 MID - NIHMS521578 EDAT- 2013/10/08 06:00 MHDA- 2014/05/03 06:00 CRDT- 2013/10/08 06:00 PHST- 2013/05/31 00:00 [received] PHST- 2013/09/04 00:00 [accepted] PHST- 2013/10/08 06:00 [entrez] PHST- 2013/10/08 06:00 [pubmed] PHST- 2014/05/03 06:00 [medline] AID - ncomms3550 [pii] AID - 10.1038/ncomms3550 [doi] PST - ppublish SO - Nat Commun. 2013;4:2550. doi: 10.1038/ncomms3550. PMID- 24052382 OWN - NLM STAT- MEDLINE DCOM- 20140402 LR - 20211021 IS - 1940-6029 (Electronic) IS - 1064-3745 (Print) IS - 1064-3745 (Linking) VI - 1071 DP - 2014 TI - Live-cell imaging of cytosolic NADH-NAD+ redox state using a genetically encoded fluorescent biosensor. PG - 83-95 LID - 10.1007/978-1-62703-622-1_7 [doi] AB - NADH is an essential redox cofactor in numerous metabolic reactions, and the cytosolic NADH-NAD(+) redox state is a key parameter in glycolysis. Conventional NADH measurements rely on chemical determination or autofluorescence imaging, which cannot assess NADH specifically in the cytosol of individual live cells. By combining a bacterial NADH-binding protein and a fluorescent protein variant, we have created a genetically encoded fluorescent biosensor of the cytosolic NADH-NAD(+) redox state, named Peredox (Hung et al., Cell Metab 14:545-554, 2011). Here, we elaborate on imaging methods and technical considerations of using Peredox to measure cytosolic NADH:NAD(+) ratios in individual live cells. FAU - Hung, Yin Pun AU - Hung YP AD - Department of Neurobiology, Harvard Medical School, Cambridge, MA, USA. FAU - Yellen, Gary AU - Yellen G LA - eng GR - DP1 EB016985/EB/NIBIB NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - United States TA - Methods Mol Biol JT - Methods in molecular biology (Clifton, N.J.) JID - 9214969 RN - 0 (Luminescent Proteins) RN - 0U46U6E8UK (NAD) RN - 33X04XA5AT (Lactic Acid) RN - 8558G7RUTR (Pyruvic Acid) SB - IM MH - Animals MH - Biosensing Techniques/*methods MH - Calibration MH - Cell Line, Tumor MH - Cell Survival MH - Cytosol/*metabolism MH - Lactic Acid/metabolism MH - Luminescent Proteins/*genetics MH - Mice MH - Molecular Imaging/*methods MH - NAD/*metabolism MH - Oxidation-Reduction MH - *Protein Engineering MH - Pyruvic Acid/metabolism MH - Single-Cell Analysis PMC - PMC4330558 MID - NIHMS660426 EDAT- 2013/09/21 06:00 MHDA- 2014/04/03 06:00 CRDT- 2013/09/21 06:00 PHST- 2013/09/21 06:00 [entrez] PHST- 2013/09/21 06:00 [pubmed] PHST- 2014/04/03 06:00 [medline] AID - 10.1007/978-1-62703-622-1_7 [doi] PST - ppublish SO - Methods Mol Biol. 2014;1071:83-95. doi: 10.1007/978-1-62703-622-1_7. PMID- 23228828 OWN - NLM STAT- MEDLINE DCOM- 20130722 LR - 20211021 IS - 1878-108X (Electronic) IS - 0166-2236 (Print) IS - 0166-2236 (Linking) VI - 36 IP - 1 DP - 2013 Jan TI - The ketogenic diet: metabolic influences on brain excitability and epilepsy. PG - 32-40 LID - S0166-2236(12)00202-0 [pii] LID - 10.1016/j.tins.2012.11.005 [doi] AB - A dietary therapy for pediatric epilepsy known as the ketogenic diet has seen a revival in its clinical use during the past decade. Although the underlying mechanism of the diet remains unknown, modern scientific approaches, such as the genetic disruption of glucose metabolism, are allowing for more detailed questions to be addressed. Recent work indicates that several mechanisms may exist for the ketogenic diet, including disruption of glutamatergic synaptic transmission, inhibition of glycolysis, and activation of ATP-sensitive potassium channels. Here, we describe on-going work in these areas that is providing a better understanding of metabolic influences on brain excitability and epilepsy. CI - Copyright © 2012 Elsevier Ltd. All rights reserved. FAU - Lutas, Andrew AU - Lutas A AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Yellen, Gary AU - Yellen G LA - eng GR - F31 NS077633/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - R56 NS072142/NS/NINDS NIH HHS/United States GR - T32 MH020017/MH/NIMH NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Review DEP - 20121208 PL - England TA - Trends Neurosci JT - Trends in neurosciences JID - 7808616 SB - IM MH - Animals MH - Brain/*metabolism MH - *Diet, Ketogenic MH - Epilepsy/*diet therapy/*metabolism MH - Humans PMC - PMC3534786 MID - NIHMS423312 EDAT- 2012/12/12 06:00 MHDA- 2013/07/23 06:00 CRDT- 2012/12/12 06:00 PHST- 2012/09/17 00:00 [received] PHST- 2012/11/13 00:00 [revised] PHST- 2012/11/13 00:00 [accepted] PHST- 2012/12/12 06:00 [entrez] PHST- 2012/12/12 06:00 [pubmed] PHST- 2013/07/23 06:00 [medline] AID - S0166-2236(12)00202-0 [pii] AID - 10.1016/j.tins.2012.11.005 [doi] PST - ppublish SO - Trends Neurosci. 2013 Jan;36(1):32-40. doi: 10.1016/j.tins.2012.11.005. Epub 2012 Dec 8. PMID- 23071265 OWN - NLM STAT- MEDLINE DCOM- 20130411 LR - 20211021 IS - 1540-7748 (Electronic) IS - 0022-1295 (Print) IS - 0022-1295 (Linking) VI - 140 IP - 5 DP - 2012 Nov TI - Charge movement in gating-locked HCN channels reveals weak coupling of voltage sensors and gate. PG - 469-79 LID - 10.1085/jgp.201210850 [doi] AB - HCN (hyperpolarization-activated cyclic nucleotide gated) pacemaker channels have an architecture similar to that of voltage-gated K(+) channels, but they open with the opposite voltage dependence. HCN channels use essentially the same positively charged voltage sensors and intracellular activation gates as K(+) channels, but apparently these two components are coupled differently. In this study, we examine the energetics of coupling between the voltage sensor and the pore by using cysteine mutant channels for which low concentrations of Cd(2+) ions freeze the open-closed gating machinery but still allow the sensors to move. We were able to lock mutant channels either into open or into closed states by the application of Cd(2+) and measure the effect on voltage sensor movement. Cd(2+) did not immobilize the gating charge, as expected for strict coupling, but rather it produced shifts in the voltage dependence of voltage sensor charge movement, consistent with its effect of confining transitions to either closed or open states. From the magnitude of the Cd(2+)-induced shifts, we estimate that each voltage sensor produces a roughly three- to sevenfold effect on the open-closed equilibrium, corresponding to a coupling energy of ∼1.3-2 kT per sensor. Such coupling is not only opposite in sign to the coupling in K(+) channels, but also much weaker. FAU - Ryu, Sujung AU - Ryu S AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 HL070320/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20121015 PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Cyclic Nucleotide-Gated Cation Channels) RN - 0 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels) RN - 0 (Potassium Channels) RN - 00BH33GNGH (Cadmium) RN - K848JZ4886 (Cysteine) RN - RWP5GA015D (Potassium) SB - IM CIN - J Gen Physiol. 2012 Nov;140(5):457-61. PMID: 23071267 MH - Animals MH - Cadmium/pharmacology MH - Cyclic Nucleotide-Gated Cation Channels/chemistry/genetics/*physiology MH - Cysteine/genetics/metabolism MH - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MH - Ion Channel Gating/drug effects/*physiology MH - Mutation, Missense MH - Potassium/metabolism MH - Potassium Channels/chemistry/genetics/*physiology MH - Protein Structure, Tertiary MH - Sea Urchins MH - Static Electricity MH - Xenopus PMC - PMC3483112 EDAT- 2012/10/17 06:00 MHDA- 2013/04/12 06:00 CRDT- 2012/10/17 06:00 PHST- 2012/10/17 06:00 [entrez] PHST- 2012/10/17 06:00 [pubmed] PHST- 2013/04/12 06:00 [medline] AID - jgp.201210850 [pii] AID - 201210850 [pii] AID - 10.1085/jgp.201210850 [doi] PST - ppublish SO - J Gen Physiol. 2012 Nov;140(5):469-79. doi: 10.1085/jgp.201210850. Epub 2012 Oct 15. PMID- 22930802 OWN - NLM STAT- MEDLINE DCOM- 20121217 LR - 20211021 IS - 1540-7748 (Electronic) IS - 0022-1295 (Print) IS - 0022-1295 (Linking) VI - 140 IP - 3 DP - 2012 Sep TI - Structural changes during HCN channel gating defined by high affinity metal bridges. PG - 279-91 LID - 10.1085/jgp.201210838 [doi] AB - Hyperpolarization-activated cyclic nucleotide-sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4-S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd(2+)-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4-S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4-S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4-S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating. FAU - Kwan, Daniel C H AU - Kwan DC AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Prole, David L AU - Prole DL FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 HL070320/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Cyclic Nucleotide-Gated Cation Channels) RN - 0 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels) RN - 0 (Potassium Channels) RN - 0 (Protein Subunits) RN - 00BH33GNGH (Cadmium) RN - K848JZ4886 (Cysteine) SB - IM MH - Amino Acid Sequence MH - Amino Acid Substitution MH - Animals MH - Cadmium/pharmacology MH - Cyclic Nucleotide-Gated Cation Channels/*chemistry/genetics/physiology MH - Cysteine/chemistry/genetics MH - HEK293 Cells MH - Humans MH - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MH - Ion Channel Gating/drug effects/*genetics MH - Models, Chemical MH - Molecular Sequence Data MH - Mutation, Missense MH - Potassium Channels/*chemistry/genetics/physiology MH - Protein Structure, Tertiary MH - Protein Subunits/chemistry MH - Strongylocentrotus purpuratus/chemistry PMC - PMC3434101 EDAT- 2012/08/30 06:00 MHDA- 2012/12/18 06:00 CRDT- 2012/08/30 06:00 PHST- 2012/08/30 06:00 [entrez] PHST- 2012/08/30 06:00 [pubmed] PHST- 2012/12/18 06:00 [medline] AID - jgp.201210838 [pii] AID - 201210838 [pii] AID - 10.1085/jgp.201210838 [doi] PST - ppublish SO - J Gen Physiol. 2012 Sep;140(3):279-91. doi: 10.1085/jgp.201210838. PMID- 22632729 OWN - NLM STAT- MEDLINE DCOM- 20120803 LR - 20220309 IS - 1097-4199 (Electronic) IS - 0896-6273 (Print) IS - 0896-6273 (Linking) VI - 74 IP - 4 DP - 2012 May 24 TI - BAD-dependent regulation of fuel metabolism and K(ATP) channel activity confers resistance to epileptic seizures. PG - 719-30 LID - 10.1016/j.neuron.2012.03.032 [doi] AB - Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phosphoregulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive K(ATP) channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the K(ATP) channel, implicating the BAD-K(ATP) axis in metabolic control of neuronal excitation and seizure responses. CI - Copyright © 2012 Elsevier Inc. All rights reserved. FAU - Giménez-Cassina, Alfredo AU - Giménez-Cassina A AD - Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. FAU - Martínez-François, Juan Ramón AU - Martínez-François JR FAU - Fisher, Jill K AU - Fisher JK FAU - Szlyk, Benjamin AU - Szlyk B FAU - Polak, Klaudia AU - Polak K FAU - Wiwczar, Jessica AU - Wiwczar J FAU - Tanner, Geoffrey R AU - Tanner GR FAU - Lutas, Andrew AU - Lutas A FAU - Yellen, Gary AU - Yellen G FAU - Danial, Nika N AU - Danial NN LA - eng GR - R01 NS055031-01A1/NS/NINDS NIH HHS/United States GR - R01 NS055031-02/NS/NINDS NIH HHS/United States GR - F31 NS077633/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - R56 NS072142-01A1/NS/NINDS NIH HHS/United States GR - T32 MH020017/MH/NIMH NIH HHS/United States GR - UL1 RR025758-03/RR/NCRR NIH HHS/United States GR - R01 NS055031-04/NS/NINDS NIH HHS/United States GR - K01CA106596/CA/NCI NIH HHS/United States GR - K01 CA106596/CA/NCI NIH HHS/United States GR - T32 GM007223/GM/NIGMS NIH HHS/United States GR - R56 NS072142/NS/NINDS NIH HHS/United States GR - UL1 RR025758/RR/NCRR NIH HHS/United States GR - K01 CA106596-05/CA/NCI NIH HHS/United States GR - R01 NS055031-03/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - United States TA - Neuron JT - Neuron JID - 8809320 RN - 0 (KATP Channels) RN - 0 (bcl-Associated Death Protein) SB - IM CIN - Neuron. 2012 May 24;74(4):600-2. PMID: 22632717 MH - Animals MH - Apoptosis/physiology MH - Astrocytes/metabolism MH - Cells, Cultured MH - Electroencephalography MH - Energy Metabolism/*physiology MH - Hippocampus/*metabolism/physiopathology MH - KATP Channels/*metabolism MH - Mice MH - Mice, Transgenic MH - Neurons/metabolism MH - Oxygen Consumption/physiology MH - Phosphorylation MH - Seizures/chemically induced/*metabolism/physiopathology MH - bcl-Associated Death Protein/genetics/*metabolism PMC - PMC3361694 MID - NIHMS370681 EDAT- 2012/05/29 06:00 MHDA- 2012/08/04 06:00 CRDT- 2012/05/29 06:00 PHST- 2012/03/08 00:00 [accepted] PHST- 2012/05/29 06:00 [entrez] PHST- 2012/05/29 06:00 [pubmed] PHST- 2012/08/04 06:00 [medline] AID - S0896-6273(12)00343-1 [pii] AID - 10.1016/j.neuron.2012.03.032 [doi] PST - ppublish SO - Neuron. 2012 May 24;74(4):719-30. doi: 10.1016/j.neuron.2012.03.032. PMID- 22341329 OWN - NLM STAT- MEDLINE DCOM- 20120716 LR - 20211021 IS - 1875-7855 (Electronic) IS - 0079-6123 (Print) IS - 0079-6123 (Linking) VI - 196 DP - 2012 TI - Optogenetic reporters: Fluorescent protein-based genetically encoded indicators of signaling and metabolism in the brain. PG - 235-63 LID - 10.1016/B978-0-444-59426-6.00012-4 [doi] AB - Fluorescent protein technology has evolved to include genetically encoded biosensors that can monitor levels of ions, metabolites, and enzyme activities as well as protein conformation and even membrane voltage. They are well suited to live-cell microscopy and quantitative analysis, and they can be used in multiple imaging modes, including one- or two-photon fluorescence intensity or lifetime microscopy. Although not nearly complete, there now exists a substantial set of genetically encoded reporters that can be used to monitor many aspects of neuronal and glial biology, and these biosensors can be used to visualize synaptic transmission and activity-dependent signaling in vitro and in vivo. In this review, we present an overview of design strategies for engineering biosensors, including sensor designs using circularly permuted fluorescent proteins and using fluorescence resonance energy transfer between fluorescent proteins. We also provide examples of indicators that sense small ions (e.g., pH, chloride, zinc), metabolites (e.g., glutamate, glucose, ATP, cAMP, lipid metabolites), signaling pathways (e.g., G protein-coupled receptors, Rho GTPases), enzyme activities (e.g., protein kinase A, caspases), and reactive species. We focus on examples where these genetically encoded indicators have been applied to brain-related studies and used with live-cell fluorescence microscopy. CI - Copyright © 2012 Elsevier B.V. All rights reserved. FAU - Tantama, Mathew AU - Tantama M AD - Department of Neurobiology, Harvard Medical School, Boston, MA, USA. FAU - Hung, Yin Pun AU - Hung YP FAU - Yellen, Gary AU - Yellen G LA - eng GR - F32 NS066613/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - F32NS066613/NS/NINDS NIH HHS/United States GR - R01NS055031/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Review PL - Netherlands TA - Prog Brain Res JT - Progress in brain research JID - 0376441 RN - 0 (Luminescent Proteins) SB - IM MH - Animals MH - Biosensing Techniques MH - Brain/*metabolism MH - Fluorescence Resonance Energy Transfer MH - Luminescent Proteins/*genetics/metabolism MH - Signal Transduction/*genetics PMC - PMC3494096 MID - NIHMS416930 EDAT- 2012/02/22 06:00 MHDA- 2012/07/17 06:00 CRDT- 2012/02/21 06:00 PHST- 2012/02/21 06:00 [entrez] PHST- 2012/02/22 06:00 [pubmed] PHST- 2012/07/17 06:00 [medline] AID - B978-0-444-59426-6.00012-4 [pii] AID - 10.1016/B978-0-444-59426-6.00012-4 [doi] PST - ppublish SO - Prog Brain Res. 2012;196:235-63. doi: 10.1016/B978-0-444-59426-6.00012-4. PMID- 21982714 OWN - NLM STAT- MEDLINE DCOM- 20120203 LR - 20220311 IS - 1932-7420 (Electronic) IS - 1550-4131 (Print) IS - 1550-4131 (Linking) VI - 14 IP - 4 DP - 2011 Oct 5 TI - Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor. PG - 545-54 LID - 10.1016/j.cmet.2011.08.012 [doi] AB - NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics. CI - Copyright © 2011 Elsevier Inc. All rights reserved. FAU - Hung, Yin Pun AU - Hung YP AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Albeck, John G AU - Albeck JG FAU - Tantama, Mathew AU - Tantama M FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 NS055031-04/NS/NINDS NIH HHS/United States GR - R01 NS055031-02/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - R01 NS055031-03/NS/NINDS NIH HHS/United States GR - F32 NS066613/NS/NINDS NIH HHS/United States GR - R01 NS055031-02S1/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. PL - United States TA - Cell Metab JT - Cell metabolism JID - 101233170 RN - 0 (Bacterial Proteins) RN - 0 (Luminescent Proteins) RN - 0 (Phosphoinositide-3 Kinase Inhibitors) RN - 0 (Recombinant Fusion Proteins) RN - 0U46U6E8UK (NAD) RN - EC 1.1.1.27 (L-Lactate Dehydrogenase) SB - IM MH - Animals MH - Bacterial Proteins/genetics/metabolism MH - *Biosensing Techniques MH - Cells, Cultured MH - Cytosol/*metabolism MH - Glycolysis MH - Hydrogen-Ion Concentration MH - L-Lactate Dehydrogenase/metabolism MH - Luminescent Proteins/genetics/*metabolism MH - Mice MH - NAD/*chemistry/metabolism MH - Oxidation-Reduction MH - Phosphatidylinositol 3-Kinases/metabolism MH - Phosphoinositide-3 Kinase Inhibitors MH - Recombinant Fusion Proteins/genetics/metabolism PMC - PMC3190165 MID - NIHMS324339 EDAT- 2011/10/11 06:00 MHDA- 2012/02/04 06:00 CRDT- 2011/10/11 06:00 PHST- 2011/03/02 00:00 [received] PHST- 2011/06/17 00:00 [revised] PHST- 2011/08/30 00:00 [accepted] PHST- 2011/10/11 06:00 [entrez] PHST- 2011/10/11 06:00 [pubmed] PHST- 2012/02/04 06:00 [medline] AID - S1550-4131(11)00342-1 [pii] AID - 10.1016/j.cmet.2011.08.012 [doi] PST - ppublish SO - Cell Metab. 2011 Oct 5;14(4):545-54. doi: 10.1016/j.cmet.2011.08.012. PMID- 21653873 OWN - NLM STAT- MEDLINE DCOM- 20110901 LR - 20211020 IS - 1529-2401 (Electronic) IS - 0270-6474 (Print) IS - 0270-6474 (Linking) VI - 31 IP - 23 DP - 2011 Jun 8 TI - Single K ATP channel opening in response to action potential firing in mouse dentate granule neurons. PG - 8689-96 LID - 10.1523/JNEUROSCI.5951-10.2011 [doi] AB - ATP-sensitive potassium channels (K(ATP) channels) are important sensors of cellular metabolic state that link metabolism and excitability in neuroendocrine cells, but their role in nonglucosensing central neurons is less well understood. To examine a possible role for K(ATP) channels in modulating excitability in hippocampal circuits, we recorded the activity of single K(ATP) channels in cell-attached patches of granule cells in the mouse dentate gyrus during bursts of action potentials generated by antidromic stimulation of the mossy fibers. Ensemble averages of the open probability (p(open)) of single K(ATP) channels over repeated trials of stimulated spike activity showed a transient increase in p(open) in response to action potential firing. Channel currents were identified as K(ATP) channels through blockade with glibenclamide and by comparison with recordings from Kir6.2 knock-out mice. The transient elevation in K(ATP) p(open) may arise from submembrane ATP depletion by the Na(+)-K(+) ATPase, as the pump blocker strophanthidin reduced the magnitude of the elevation. Both the steady-state and stimulus-elevated p(open) of the recorded channels were higher in the presence of the ketone body R-β-hydroxybutyrate, consistent with earlier findings that ketone bodies can affect K(ATP) activity. Using perforated-patch recording, we also found that K(ATP) channels contribute to the slow afterhyperpolarization following an evoked burst of action potentials. We propose that activity-dependent opening of K(ATP) channels may help granule cells act as a seizure gate in the hippocampus and that ketone-body-mediated augmentation of the activity-dependent opening could in part explain the effect of the ketogenic diet in reducing epileptic seizures. FAU - Tanner, Geoffrey R AU - Tanner GR AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA. FAU - Lutas, Andrew AU - Lutas A FAU - Martínez-François, Juan Ramón AU - Martínez-François JR FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 NS055031-04/NS/NINDS NIH HHS/United States GR - NS029693/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - RR025758/RR/NCRR NIH HHS/United States GR - UL1 RR025758/RR/NCRR NIH HHS/United States GR - R01 NS029693/NS/NINDS NIH HHS/United States GR - UL1 RR025758-03/RR/NCRR NIH HHS/United States GR - NS055031/NS/NINDS NIH HHS/United States GR - R01 NS029693-17/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - United States TA - J Neurosci JT - The Journal of neuroscience : the official journal of the Society for Neuroscience JID - 8102140 RN - 0 (KATP Channels) SB - IM MH - Action Potentials/*physiology MH - Animals MH - Dentate Gyrus/*physiology MH - Electrophysiology MH - Ion Channel Gating/physiology MH - KATP Channels/*physiology MH - Membrane Potentials/physiology MH - Mice MH - Neurons/*physiology PMC - PMC3133530 MID - NIHMS302797 EDAT- 2011/06/10 06:00 MHDA- 2011/09/02 06:00 CRDT- 2011/06/10 06:00 PHST- 2011/06/10 06:00 [entrez] PHST- 2011/06/10 06:00 [pubmed] PHST- 2011/09/02 06:00 [medline] AID - 31/23/8689 [pii] AID - 3703814 [pii] AID - 10.1523/JNEUROSCI.5951-10.2011 [doi] PST - ppublish SO - J Neurosci. 2011 Jun 8;31(23):8689-96. doi: 10.1523/JNEUROSCI.5951-10.2011. PMID- 21631110 OWN - NLM STAT- MEDLINE DCOM- 20120820 LR - 20231213 IS - 1520-5126 (Electronic) IS - 0002-7863 (Print) IS - 0002-7863 (Linking) VI - 133 IP - 26 DP - 2011 Jul 6 TI - Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor. PG - 10034-7 LID - 10.1021/ja202902d [doi] AB - Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH. FAU - Tantama, Mathew AU - Tantama M AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA. FAU - Hung, Yin Pun AU - Hung YP FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 NS055031-04/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - F32 NS066613/NS/NINDS NIH HHS/United States GR - R01NS055031/NS/NINDS NIH HHS/United States GR - F32 NS066613-01A2/NS/NINDS NIH HHS/United States GR - F32NS066613/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20110609 PL - United States TA - J Am Chem Soc JT - Journal of the American Chemical Society JID - 7503056 RN - 0 (Luminescent Proteins) SB - IM MH - Animals MH - Cell Line MH - Cell Survival MH - Genetic Engineering/*methods MH - Hydrogen-Ion Concentration MH - Intracellular Space/*chemistry/metabolism MH - Luminescent Proteins/*genetics MH - Mice MH - Molecular Imaging/*methods MH - Mutagenesis MH - Spectrometry, Fluorescence MH - Red Fluorescent Protein PMC - PMC3126897 MID - NIHMS301322 EDAT- 2011/06/03 06:00 MHDA- 2012/08/21 06:00 CRDT- 2011/06/03 06:00 PHST- 2011/06/03 06:00 [entrez] PHST- 2011/06/03 06:00 [pubmed] PHST- 2012/08/21 06:00 [medline] AID - 10.1021/ja202902d [doi] PST - ppublish SO - J Am Chem Soc. 2011 Jul 6;133(26):10034-7. doi: 10.1021/ja202902d. Epub 2011 Jun 9. PMID- 19122669 OWN - NLM STAT- MEDLINE DCOM- 20090223 LR - 20211020 IS - 1548-7105 (Electronic) IS - 1548-7091 (Print) IS - 1548-7091 (Linking) VI - 6 IP - 2 DP - 2009 Feb TI - A genetically encoded fluorescent reporter of ATP:ADP ratio. PG - 161-6 LID - 10.1038/nmeth.1288 [doi] AB - We constructed a fluorescent sensor of adenylate nucleotides by combining a circularly permuted variant of GFP with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The sensor's affinity for Mg-ATP was <100 nM, as seen for other members of the bacterial PII regulator family, a surprisingly high affinity given that normal intracellular ATP concentration is in the millimolar range. ADP bound the same site of the sensor as Mg-ATP, competing with it, but produced a smaller change in fluorescence. At physiological ATP and ADP concentrations, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP:ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high-affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP:ADP ratio during live-cell imaging. FAU - Berg, Jim AU - Berg J AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, Massachusetts 02115, USA. FAU - Hung, Yin Pun AU - Hung YP FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 NS055031-01A1/NS/NINDS NIH HHS/United States GR - NS029693/NS/NINDS NIH HHS/United States GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - F31 NS048798/NS/NINDS NIH HHS/United States GR - R01 NS029693/NS/NINDS NIH HHS/United States GR - NS055031/NS/NINDS NIH HHS/United States GR - R01 NS029693-17/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20090104 PL - United States TA - Nat Methods JT - Nature methods JID - 101215604 RN - 0 (Archaeal Proteins) RN - 0 (Bacterial Proteins) RN - 0 (DNA, Bacterial) RN - 0 (Fluorescent Dyes) RN - 0 (Luminescent Proteins) RN - 0 (yellow fluorescent protein, Bacteria) RN - 61D2G4IYVH (Adenosine Diphosphate) RN - 8L70Q75FXE (Adenosine Triphosphate) SB - IM MH - Adenosine Diphosphate/*analysis MH - Adenosine Triphosphate/*analysis MH - Archaeal Proteins/*chemistry MH - Bacterial Proteins/*chemistry/genetics MH - Binding Sites MH - Biosensing Techniques/methods MH - DNA, Bacterial/chemistry/genetics MH - Fluorescent Dyes/*chemistry MH - Luminescent Proteins/*chemistry/genetics MH - Microscopy, Fluorescence MH - Polymerase Chain Reaction PMC - PMC2633436 MID - NIHMS80615 EDAT- 2009/01/06 09:00 MHDA- 2009/02/24 09:00 CRDT- 2009/01/06 09:00 PHST- 2008/07/12 00:00 [received] PHST- 2008/11/17 00:00 [accepted] PHST- 2009/01/06 09:00 [entrez] PHST- 2009/01/06 09:00 [pubmed] PHST- 2009/02/24 09:00 [medline] AID - nmeth.1288 [pii] AID - 10.1038/nmeth.1288 [doi] PST - ppublish SO - Nat Methods. 2009 Feb;6(2):161-6. doi: 10.1038/nmeth.1288. Epub 2009 Jan 4. PMID- 19049596 OWN - NLM STAT- MEDLINE DCOM- 20090122 LR - 20211020 IS - 1528-1167 (Electronic) IS - 0013-9580 (Print) IS - 0013-9580 (Linking) VI - 49 Suppl 8 IP - Suppl 8 DP - 2008 Nov TI - Ketone bodies, glycolysis, and KATP channels in the mechanism of the ketogenic diet. PG - 80-2 LID - 10.1111/j.1528-1167.2008.01843.x [doi] AB - The ketogenic diet (KD) has shown remarkable efficacy in the treatment of drug-resistant childhood epilepsy. Our understanding of how the KD produces its anticonvulsant and antiepileptogenic effects remains incomplete, which is perhaps not surprising for a biological manipulation as sweeping as dietary change. Several hypotheses focus on ketone bodies, fuel molecules that circulate at millimolar concentrations in the blood of patients on a KD, as causative agents. Here I consider some recent evidence for one such hypothesis, involving a possible role for altered glycolysis and consequent activation of a class of potassium channels called K(ATP)channels. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA. gary yellen@hms.harvard.edu LA - eng GR - R01 NS055031/NS/NINDS NIH HHS/United States GR - R01 NS055031-01A1/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Review PL - United States TA - Epilepsia JT - Epilepsia JID - 2983306R RN - 0 (KATP Channels) RN - 0 (Ketone Bodies) SB - IM MH - Animals MH - Brain/metabolism MH - *Diet, Ketogenic MH - Epilepsy/*diet therapy MH - Glycolysis/*physiology MH - Humans MH - KATP Channels/*metabolism MH - Ketone Bodies/*metabolism PMC - PMC2646251 MID - NIHMS92452 COIS- The author of this article discloses that there are no conflicts of interest. EDAT- 2008/12/17 09:00 MHDA- 2009/01/23 09:00 CRDT- 2008/12/17 09:00 PHST- 2008/12/17 09:00 [pubmed] PHST- 2009/01/23 09:00 [medline] PHST- 2008/12/17 09:00 [entrez] AID - EPI1843 [pii] AID - 10.1111/j.1528-1167.2008.01843.x [doi] PST - ppublish SO - Epilepsia. 2008 Nov;49 Suppl 8(Suppl 8):80-2. doi: 10.1111/j.1528-1167.2008.01843.x. PMID- 17409226 OWN - NLM STAT- MEDLINE DCOM- 20070502 LR - 20200225 IS - 1529-2401 (Electronic) IS - 0270-6474 (Print) IS - 0270-6474 (Linking) VI - 27 IP - 14 DP - 2007 Apr 4 TI - Ketogenic diet metabolites reduce firing in central neurons by opening K(ATP) channels. PG - 3618-25 AB - A low-carbohydrate ketogenic diet remains one of the most effective (but mysterious) treatments for severe pharmacoresistant epilepsy. We have tested for an acute effect of physiological ketone bodies on neuronal firing rates and excitability, to discover possible therapeutic mechanisms of the ketogenic diet. Physiological concentrations of ketone bodies (beta-hydroxybutyrate or acetoacetate) reduced the spontaneous firing rate of neurons in slices from rat or mouse substantia nigra pars reticulata. This region is thought to act as a "seizure gate," controlling seizure generalization. Consistent with an anticonvulsant role, the ketone body effect is larger for cells that fire more rapidly. The effect of ketone bodies was abolished by eliminating the metabolically sensitive K(ATP) channels pharmacologically or by gene knock-out. We propose that ketone bodies or glycolytic restriction treat epilepsy by augmenting a natural activity-limiting function served by K(ATP) channels in neurons. FAU - Ma, Weiyuan AU - Ma W AD - Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA. FAU - Berg, Jim AU - Berg J FAU - Yellen, Gary AU - Yellen G LA - eng GR - F31 NS048798/NS/NINDS NIH HHS/United States PT - Comparative Study PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - United States TA - J Neurosci JT - The Journal of neuroscience : the official journal of the Society for Neuroscience JID - 8102140 RN - 0 (Dietary Carbohydrates) RN - 0 (Ketone Bodies) RN - 0 (Ketones) RN - 0 (Potassium Channels) RN - 8L70Q75FXE (Adenosine Triphosphate) SB - IM CIN - Epilepsy Curr. 2007 Sep-Oct;7(5):142-4. PMID: 17998978 MH - Action Potentials/*physiology MH - Adenosine Triphosphate/*metabolism MH - Animals MH - Diet, Carbohydrate-Restricted MH - Dietary Carbohydrates/*metabolism MH - Ion Channel Gating/physiology MH - Ketone Bodies/*metabolism MH - Ketones/metabolism MH - Mice MH - Mice, Knockout MH - Neurons/*metabolism MH - Potassium Channels/*metabolism MH - Rats MH - Rats, Sprague-Dawley PMC - PMC6672398 EDAT- 2007/04/06 09:00 MHDA- 2007/05/03 09:00 CRDT- 2007/04/06 09:00 PHST- 2007/04/06 09:00 [pubmed] PHST- 2007/05/03 09:00 [medline] PHST- 2007/04/06 09:00 [entrez] AID - 27/14/3618 [pii] AID - 3205442 [pii] AID - 10.1523/JNEUROSCI.0132-07.2007 [doi] PST - ppublish SO - J Neurosci. 2007 Apr 4;27(14):3618-25. doi: 10.1523/JNEUROSCI.0132-07.2007. PMID- 17043149 OWN - NLM STAT- MEDLINE DCOM- 20061228 LR - 20181113 IS - 0022-1295 (Print) IS - 1540-7748 (Electronic) IS - 0022-1295 (Linking) VI - 128 IP - 5 DP - 2006 Nov TI - Cooperative gating between single HCN pacemaker channels. PG - 561-7 AB - HCN pacemaker channels (I(f), I(q), or I(h)) play a fundamental role in the physiology of many excitable cell types, including cardiac myocytes and central neurons. While cloned HCN channels have been studied extensively in macroscopic patch clamp experiments, their extremely small conductance has precluded single channel analysis to date. Nevertheless, there remain fundamental questions about HCN gating that can be resolved only at the single channel level. Here we present the first detailed single channel study of cloned mammalian HCN2. Excised patch clamp recordings revealed discrete hyperpolarization-activated, cAMP-sensitive channel openings with amplitudes of 150-230 fA in the activation voltage range. The average conductance of these openings was approximately 1.5 pS at -120 mV in symmetrical 160 mM K(+). Some traces with multiple channels showed unusual gating behavior, characterized by a variable long delay after a voltage step followed by runs of openings. Noise analysis on macroscopic currents revealed fluctuations whose magnitudes were systematically larger than predicted from the actual single channel current size, consistent with cooperativity between single HCN channels. FAU - Dekker, John P AU - Dekker JP AD - Department of Neurobiology Harvard Medical School, Boston, MA 02115, USA. FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 HL070320/HL/NHLBI NIH HHS/United States GR - HL70320/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20061016 PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (HCN2 protein, human) RN - 0 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels) RN - 0 (Ion Channels) RN - 0 (Potassium Channels) RN - E0399OZS9N (Cyclic AMP) SB - IM MH - Cell Line MH - Cyclic AMP/physiology MH - Electrophysiology MH - Humans MH - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MH - Ion Channel Gating/*physiology MH - Ion Channels/genetics/*physiology MH - Kidney/cytology/embryology MH - Patch-Clamp Techniques MH - Potassium Channels PMC - PMC2151591 EDAT- 2006/10/18 09:00 MHDA- 2006/12/29 09:00 CRDT- 2006/10/18 09:00 PHST- 2006/10/18 09:00 [pubmed] PHST- 2006/12/29 09:00 [medline] PHST- 2006/10/18 09:00 [entrez] AID - jgp.200609599 [pii] AID - 200609599 [pii] AID - 10.1085/jgp.200609599 [doi] PST - ppublish SO - J Gen Physiol. 2006 Nov;128(5):561-7. doi: 10.1085/jgp.200609599. Epub 2006 Oct 16. PMID- 16908727 OWN - NLM STAT- MEDLINE DCOM- 20061127 LR - 20181113 IS - 0022-1295 (Print) IS - 1540-7748 (Electronic) IS - 0022-1295 (Linking) VI - 128 IP - 3 DP - 2006 Sep TI - Reversal of HCN channel voltage dependence via bridging of the S4-S5 linker and Post-S6. PG - 273-82 AB - Voltage-gated ion channels possess charged domains that move in response to changes in transmembrane voltage. How this movement is transduced into gating of the channel pore is largely unknown. Here we show directly that two functionally important regions of the spHCN1 pacemaker channel, the S4-S5 linker and the C-linker, come into close proximity during gating. Cross-linking these regions with high-affinity metal bridges or disulfide bridges dramatically alters channel gating in the absence of cAMP; after modification the polarity of voltage dependence is reversed. Instead of being closed at positive voltage and activating with hyperpolarization, modified channels are closed at negative voltage and activate with depolarization. Mechanistically, this reversal of voltage dependence occurs as a result of selectively eliminating channel deactivation, while retaining an existing inactivation process. Bridging also alters channel activation by cAMP, showing that interaction of these two regions can also affect the efficacy of physiological ligands. FAU - Prole, David L AU - Prole DL AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 HL070320/HL/NHLBI NIH HHS/United States GR - HL70320/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20060814 PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Cyclic Nucleotide-Gated Cation Channels) RN - 0 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels) RN - 0 (Ion Channels) RN - 0 (Potassium Channels) RN - 0 (Protein Subunits) RN - E0399OZS9N (Cyclic AMP) RN - K848JZ4886 (Cysteine) SB - IM MH - Amino Acid Sequence MH - Animals MH - Cells, Cultured MH - Cyclic AMP/metabolism MH - Cyclic Nucleotide-Gated Cation Channels MH - Cysteine/genetics/metabolism MH - Humans MH - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MH - Ion Channel Gating/*physiology MH - Ion Channels/*chemistry/genetics/metabolism/physiology MH - Membrane Potentials MH - Models, Biological MH - Molecular Sequence Data MH - Point Mutation MH - Potassium Channels MH - Protein Structure, Secondary MH - Protein Subunits/physiology MH - Sea Urchins/metabolism PMC - PMC2151568 EDAT- 2006/08/16 09:00 MHDA- 2006/12/09 09:00 CRDT- 2006/08/16 09:00 PHST- 2006/08/16 09:00 [pubmed] PHST- 2006/12/09 09:00 [medline] PHST- 2006/08/16 09:00 [entrez] AID - jgp.200609590 [pii] AID - 200609590 [pii] AID - 10.1085/jgp.200609590 [doi] PST - ppublish SO - J Gen Physiol. 2006 Sep;128(3):273-82. doi: 10.1085/jgp.200609590. Epub 2006 Aug 14. PMID- 16446506 OWN - NLM STAT- MEDLINE DCOM- 20060404 LR - 20220309 IS - 0022-1295 (Print) IS - 1540-7748 (Electronic) IS - 0022-1295 (Linking) VI - 127 IP - 2 DP - 2006 Feb TI - Distinct populations of HCN pacemaker channels produce voltage-dependent and voltage-independent currents. PG - 183-90 AB - Hyperpolarization-activated HCN pacemaker channels are critical for the generation of spontaneous activity and the regulation of excitability in the heart and in many types of neurons. These channels produce both a voltage-dependent current (I(h)) and a voltage-independent current (I(inst) or VIC). In this study, we explored the molecular basis of the voltage-independent current. We found that for the spHCN isoform, VIC averaged approximately 4% of the maximum HCN conductance that could be activated by hyperpolarization. Cyclic AMP increased the voltage-independent current in spHCN to approximately 8% of maximum. In HCN2, VIC was approximately 2% of the maximal current, and was little affected by cAMP. VIC in both spHCN and HCN2 was blocked rapidly both by ZD7288 (an HCN channel blocker that is thought to bind in the conduction pore) and by application of Cd2+ to channels containing an introduced cysteine in the pore (spHCN-464C or HCN2-436C). These results suggest that VIC flows through the main conduction pathway, down the central axis of the protein. We suspected that VIC simply represented a nonzero limiting open probability for HCN channels at positive voltages. Surprisingly, we found instead that the spHCN channels carrying VIC were not in rapid equilibrium with the channels carrying the voltage-dependent current, because they could be blocked independently; a single application of blocker at a depolarized potential essentially eliminated VIC with little change in I(h). Thus, VIC appears to be produced by a distinct population of HCN channels. This voltage-independent current could contribute significantly to the role of HCN channels in neurons and myocytes; VIC flowing through the channels at physiological potentials would tend to promote excitability by accelerating both depolarization and repolarization. FAU - Proenza, Catherine AU - Proenza C AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Yellen, Gary AU - Yellen G LA - eng GR - F32 HL071365/HL/NHLBI NIH HHS/United States GR - R01 HL070320/HL/NHLBI NIH HHS/United States GR - HL70320/HL/NHLBI NIH HHS/United States GR - HL71365/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Cyclic Nucleotide-Gated Cation Channels) RN - 0 (HCN2 protein, human) RN - 0 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels) RN - 0 (Ion Channels) RN - 0 (Potassium Channels) RN - 0 (Protein Isoforms) RN - 0 (Pyrimidines) RN - 00BH33GNGH (Cadmium) RN - 133059-99-1 (ICI D2788) RN - E0399OZS9N (Cyclic AMP) SB - IM MH - Cadmium/metabolism MH - Cell Line MH - Cyclic AMP/pharmacology MH - Cyclic Nucleotide-Gated Cation Channels MH - Electric Conductivity MH - Humans MH - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MH - Ion Channel Gating MH - Ion Channels/antagonists & inhibitors/*chemistry/*physiology MH - *Membrane Potentials MH - Myocytes, Cardiac/physiology MH - Neurons/physiology MH - Patch-Clamp Techniques MH - Potassium Channels MH - Protein Isoforms/chemistry/physiology MH - Pyrimidines/pharmacology MH - Time Factors PMC - PMC2151495 EDAT- 2006/02/01 09:00 MHDA- 2006/04/06 09:00 CRDT- 2006/02/01 09:00 PHST- 2006/02/01 09:00 [pubmed] PHST- 2006/04/06 09:00 [medline] PHST- 2006/02/01 09:00 [entrez] AID - jgp.200509389 [pii] AID - 200509389 [pii] AID - 10.1085/jgp.200509389 [doi] PST - ppublish SO - J Gen Physiol. 2006 Feb;127(2):183-90. doi: 10.1085/jgp.200509389. PMID- 16260836 OWN - NLM STAT- MEDLINE DCOM- 20060210 LR - 20181113 IS - 0022-1295 (Print) IS - 1540-7748 (Electronic) IS - 0022-1295 (Linking) VI - 126 IP - 5 DP - 2005 Nov TI - Status of the intracellular gate in the activated-not-open state of shaker K+ channels. PG - 419-28 AB - Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421-439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389-414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore. FAU - del Camino, Donato AU - del Camino D AD - Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. FAU - Kanevsky, Max AU - Kanevsky M FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 NS029693/NS/NINDS NIH HHS/United States GR - NS 29693/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Potassium Channel Blockers) RN - 0 (Shaker Superfamily of Potassium Channels) RN - 00BH33GNGH (Cadmium) RN - 66-40-0 (Tetraethylammonium) RN - BH3B64OKL9 (4-Aminopyridine) RN - K848JZ4886 (Cysteine) SB - IM MH - 4-Aminopyridine/metabolism/pharmacology MH - Animals MH - Binding Sites MH - Cadmium/metabolism/pharmacology MH - Cell Membrane Permeability MH - Cysteine/chemistry/genetics MH - Ion Channel Gating/drug effects/*physiology MH - Ion Transport/drug effects/physiology MH - Membrane Potentials/drug effects/physiology MH - Mutagenesis MH - Oocytes MH - Potassium Channel Blockers/pharmacology MH - Protein Conformation MH - Shaker Superfamily of Potassium Channels/chemistry/drug effects/*physiology MH - Tetraethylammonium/metabolism/pharmacology MH - Xenopus laevis/physiology PMC - PMC1794167 MID - NIHMS6157 EDAT- 2005/11/02 09:00 MHDA- 2006/02/14 09:00 CRDT- 2005/11/02 09:00 PHST- 2005/11/02 09:00 [pubmed] PHST- 2006/02/14 09:00 [medline] PHST- 2005/11/02 09:00 [entrez] AID - jgp.200509385 [pii] AID - 200509385 [pii] AID - 10.1085/jgp.200509385 [doi] PST - ppublish SO - J Gen Physiol. 2005 Nov;126(5):419-28. doi: 10.1085/jgp.200509385. PMID- 15103379 OWN - NLM STAT- MEDLINE DCOM- 20040504 LR - 20161025 IS - 1476-4687 (Electronic) IS - 0028-0836 (Linking) VI - 428 IP - 6985 DP - 2004 Apr 22 TI - Intracellular gate opening in Shaker K+ channels defined by high-affinity metal bridges. PG - 864-8 AB - Voltage-gated potassium channels such as Shaker help to control electrical signalling in neurons by regulating the passage of K+ across cell membranes. Ion flow is controlled by a voltage-dependent gate at the intracellular side of the pore, formed by the crossing of four alpha-helices--the inner-pore helices. The prevailing model of gating is based on a comparison of the crystal structures of two bacterial channels--KcsA in a closed state and MthK in an open state--and proposes a hinge motion at a conserved glycine that splays the inner-pore helices wide open. We show here that two types of intersubunit metal bridge, involving cysteines placed near the bundle crossing, can occur simultaneously in the open state. These bridges provide constraints on the open Shaker channel structure, and on the degree of movement upon opening. We conclude that, unlike predictions from the structure of MthK, the inner-pore helices of Shaker probably maintain the KcsA-like bundle-crossing motif in the open state, with a bend in this region at the conserved proline motif (Pro-X-Pro) not found in the bacterial channels. A narrower opening of the bundle crossing in Shaker K+ channels may help to explain why Shaker has an approximately tenfold lower conductance than its bacterial relatives. FAU - Webster, Sarah M AU - Webster SM AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115, USA. FAU - Del Camino, Donato AU - Del Camino D FAU - Dekker, John P AU - Dekker JP FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 NS029693/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, U.S. Gov't, P.H.S. PL - England TA - Nature JT - Nature JID - 0410462 RN - 0 (Bacterial Proteins) RN - 0 (Ligands) RN - 0 (Potassium Channels) RN - 0 (Shaker Superfamily of Potassium Channels) RN - 0 (prokaryotic potassium channel) RN - 00BH33GNGH (Cadmium) RN - RWP5GA015D (Potassium) SB - IM MH - Bacterial Proteins/chemistry/genetics/metabolism MH - Cadmium/*metabolism MH - Cell Line MH - Electric Conductivity MH - Humans MH - *Ion Channel Gating MH - Ion Transport MH - Ligands MH - Models, Molecular MH - Mutation MH - Potassium/metabolism MH - Potassium Channels/chemistry/genetics/*metabolism MH - Protein Structure, Quaternary MH - Shaker Superfamily of Potassium Channels EDAT- 2004/04/23 05:00 MHDA- 2004/05/05 05:00 CRDT- 2004/04/23 05:00 PHST- 2003/11/20 00:00 [received] PHST- 2004/03/04 00:00 [accepted] PHST- 2004/04/23 05:00 [pubmed] PHST- 2004/05/05 05:00 [medline] PHST- 2004/04/23 05:00 [entrez] AID - nature02468 [pii] AID - 10.1038/nature02468 [doi] PST - ppublish SO - Nature. 2004 Apr 22;428(6985):864-8. doi: 10.1038/nature02468. PMID- 15003173 OWN - NLM STAT- MEDLINE DCOM- 20040412 LR - 20190823 IS - 0896-6273 (Print) IS - 0896-6273 (Linking) VI - 41 IP - 5 DP - 2004 Mar 4 TI - Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage. PG - 737-44 AB - Hyperpolarization-activated HCN channels are modulated by direct binding of cyclic nucleotides. For HCN2 channels, cAMP shifts the voltage dependence for activation, with relatively little change in the maximal conductance. By contrast, in spHCN channels, cAMP relieves a rapid inactivation process and produces a large increase in maximum conductance. Our results suggest that these two effects of cAMP represent the same underlying process. We also find that spHCN inactivation occurs not by closure of a specialized inactivation gate, as for other voltage-dependent channels, but by reclosure of the same intracellular gate opened upon activation. Effectively, the activation gate exhibits a "desensitization to voltage," perhaps by slippage of the coupling between the voltage sensors and the gate. Differences in the initial coupling efficiency could allow cAMP to produce either the inactivation or the shift phenotype by strengthening effective coupling: a shift would naturally occur if coupling is already strong in the absence of cAMP. FAU - Shin, Ki Soon AU - Shin KS AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115 USA. FAU - Maertens, Chantal AU - Maertens C FAU - Proenza, Catherine AU - Proenza C FAU - Rothberg, Brad S AU - Rothberg BS FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 HL070320/HL/NHLBI NIH HHS/United States GR - HL70320/HL/NHLBI NIH HHS/United States GR - HL71365/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - Neuron JT - Neuron JID - 8809320 RN - 0 (HCN2 protein, human) RN - 0 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels) RN - 0 (Ion Channels) RN - 0 (Muscle Proteins) RN - 0 (Potassium Channels) RN - E0399OZS9N (Cyclic AMP) SB - IM MH - Binding Sites/physiology MH - Cell Line MH - Cyclic AMP/pharmacology MH - Humans MH - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MH - Ion Channel Gating/*physiology MH - Ion Channels/*antagonists & inhibitors/chemistry/*metabolism MH - Membrane Potentials/physiology MH - Muscle Proteins/*antagonists & inhibitors/chemistry/*metabolism MH - Potassium Channels EDAT- 2004/03/09 05:00 MHDA- 2004/04/13 05:00 CRDT- 2004/03/09 05:00 PHST- 2003/10/30 00:00 [received] PHST- 2004/01/05 00:00 [revised] PHST- 2004/01/06 00:00 [accepted] PHST- 2004/03/09 05:00 [pubmed] PHST- 2004/04/13 05:00 [medline] PHST- 2004/03/09 05:00 [entrez] AID - S0896627304000832 [pii] AID - 10.1016/s0896-6273(04)00083-2 [doi] PST - ppublish SO - Neuron. 2004 Mar 4;41(5):737-44. doi: 10.1016/s0896-6273(04)00083-2. PMID- 14962924 OWN - NLM STAT- MEDLINE DCOM- 20050106 LR - 20191210 IS - 1367-4803 (Print) IS - 1367-4803 (Linking) VI - 20 IP - 10 DP - 2004 Jul 10 TI - A perturbation-based method for calculating explicit likelihood of evolutionary co-variance in multiple sequence alignments. PG - 1565-72 AB - MOTIVATION: The constituent amino acids of a protein work together to define its structure and to facilitate its function. Their interdependence should be apparent in the evolutionary record of each protein family: positions in the sequence of a protein family that are intimately associated in space or in function should co-vary in evolution. A recent approach by Ranganathan and colleagues proposes to look at subsets of a protein family, selected for their sequence at one position, to see how this affects variation at other positions. RESULTS: We present a quantitative algorithm for assessing covariation with this approach, based on explicit likelihood calculations. By applying our algorithm to 138 Pfam families with at least one member of known structure, we demonstrate that our method has improved power in finding physically close residues in crystal structures, compared to that of Ranganathan and colleagues. SUPPLEMENTARY INFORMATION: www.afodor.net/bioinfosup.html FAU - Dekker, John P AU - Dekker JP AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA. FAU - Fodor, Anthony AU - Fodor A FAU - Aldrich, Richard W AU - Aldrich RW FAU - Yellen, Gary AU - Yellen G LA - eng PT - Comparative Study PT - Evaluation Study PT - Journal Article PT - Validation Study DEP - 20040212 PL - England TA - Bioinformatics JT - Bioinformatics (Oxford, England) JID - 9808944 SB - IM MH - *Algorithms MH - Computer Simulation MH - *Evolution, Molecular MH - Genetic Variation MH - Likelihood Functions MH - Models, Genetic MH - Models, Statistical MH - Regression Analysis MH - Sequence Alignment/*methods MH - Sequence Analysis, Protein/*methods EDAT- 2004/02/14 05:00 MHDA- 2005/01/07 09:00 CRDT- 2004/02/14 05:00 PHST- 2004/02/14 05:00 [pubmed] PHST- 2005/01/07 09:00 [medline] PHST- 2004/02/14 05:00 [entrez] AID - bth128 [pii] AID - 10.1093/bioinformatics/bth128 [doi] PST - ppublish SO - Bioinformatics. 2004 Jul 10;20(10):1565-72. doi: 10.1093/bioinformatics/bth128. Epub 2004 Feb 12. PMID- 14557404 OWN - NLM STAT- MEDLINE DCOM- 20040629 LR - 20181113 IS - 0022-1295 (Print) IS - 1540-7748 (Electronic) IS - 0022-1295 (Linking) VI - 122 IP - 5 DP - 2003 Nov TI - Movements near the gate of a hyperpolarization-activated cation channel. PG - 501-10 AB - Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Like the related depolarization-activated K+ channels (Kv channels), HCN channels use an intracellular activation gate to regulate access to an inner cavity, lined by the S6 transmembrane regions, which leads to the selectivity filter near the extracellular surface. Here we describe two types of metal interactions with substituted cysteines in the S6, which alter the voltage-controlled movements of the gate. At one position (L466), substitution of cysteine in all four subunits allows Cd2+ ions at nanomolar concentration to stabilize the open state (a "lock-open" effect). This effect depends on native histidines at a nearby position (H462); the lock-open effect can be abolished by changing the histidines to tyrosines, or enhanced by changing them to cysteines. Unlike a similar effect in Kv channels, this effect depends on a Cd2+ bridge between 462 and 466 in the same subunit. Cysteine substitution at another position (Q468) produces two effects of Cd2+: both a lock-open effect and a dramatic slowing of channel activation-a "lock-closed" effect. The two effects can be separated, because the lock-open effect depends on the histidine at position 462. The novel lock-closed effect results from stabilization of the closed state by the binding of up to four Cd2+ ions. During the opening conformational change, the S6 apparently moves from one position in which the 468C cysteines can bind four Cd2+ ions, possibly as a cluster of cysteines and cadmium ions near the central axis of the pore, to another position (or flexible range of positions) where either 466C or 468C can bind Cd2+ in association with the histidine at 462. FAU - Rothberg, Brad S AU - Rothberg BS AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115. email: Gary_Yellen@hms.harvard.edu FAU - Shin, Ki Soon AU - Shin KS FAU - Yellen, Gary AU - Yellen G LA - eng GR - R01 HL070320/HL/NHLBI NIH HHS/United States GR - HL70320/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, P.H.S. DEP - 20031013 PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Cyclic Nucleotide-Gated Cation Channels) RN - 0 (HCN1 protein, human) RN - 0 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels) RN - 0 (Ion Channels) RN - 0 (Potassium Channels) RN - 00BH33GNGH (Cadmium) SB - IM MH - Amino Acid Sequence/physiology MH - Cadmium/metabolism/pharmacology MH - Cell Line MH - Cyclic Nucleotide-Gated Cation Channels MH - Humans MH - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MH - Ion Channel Gating/drug effects/*physiology MH - Ion Channels/genetics/*metabolism MH - Molecular Sequence Data MH - Mutation/physiology MH - Potassium Channels MH - Protein Binding/drug effects/physiology PMC - PMC2229576 EDAT- 2003/10/15 05:00 MHDA- 2004/06/30 05:00 CRDT- 2003/10/15 05:00 PHST- 2003/10/15 05:00 [pubmed] PHST- 2004/06/30 05:00 [medline] PHST- 2003/10/15 05:00 [entrez] AID - jgp.200308928 [pii] AID - 200308928 [pii] AID - 10.1085/jgp.200308928 [doi] PST - ppublish SO - J Gen Physiol. 2003 Nov;122(5):501-10. doi: 10.1085/jgp.200308928. Epub 2003 Oct 13. PMID- 12214225 OWN - NLM STAT- MEDLINE DCOM- 20021004 LR - 20220331 IS - 0028-0836 (Print) IS - 0028-0836 (Linking) VI - 419 IP - 6902 DP - 2002 Sep 5 TI - The voltage-gated potassium channels and their relatives. PG - 35-42 AB - The voltage-gated potassium channels are the prototypical members of a family of membrane signalling proteins. These protein-based machines have pores that pass millions of ions per second across the membrane with astonishing selectivity, and their gates snap open and shut in milliseconds as they sense changes in voltage or ligand concentration. The architectural modules and functional components of these sophisticated signalling molecules are becoming clear, but some important links remain to be elucidated. FAU - Yellen, Gary AU - Yellen G AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115, USA. gary_yellen@hms.harvard.edu LA - eng GR - R01 NS029693/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, U.S. Gov't, P.H.S. PT - Review PL - England TA - Nature JT - Nature JID - 0410462 RN - 0 (Ligands) RN - 0 (Potassium Channels, Voltage-Gated) RN - 9NEZ333N27 (Sodium) RN - RWP5GA015D (Potassium) RN - SY7Q814VUP (Calcium) SB - IM MH - Animals MH - Calcium/metabolism MH - Electric Conductivity MH - *Ion Channel Gating MH - Ligands MH - Models, Molecular MH - Potassium/metabolism MH - Potassium Channels, Voltage-Gated/*chemistry/*metabolism MH - Protein Conformation MH - Signal Transduction MH - Sodium/metabolism MH - Substrate Specificity RF - 81 EDAT- 2002/09/06 10:00 MHDA- 2002/10/09 04:00 CRDT- 2002/09/06 10:00 PHST- 2002/09/06 10:00 [pubmed] PHST- 2002/10/09 04:00 [medline] PHST- 2002/09/06 10:00 [entrez] AID - nature00978 [pii] AID - 10.1038/nature00978 [doi] PST - ppublish SO - Nature. 2002 Sep 5;419(6902):35-42. doi: 10.1038/nature00978. PMID- 11865022 OWN - NLM STAT- MEDLINE DCOM- 20020624 LR - 20190508 IS - 0022-1295 (Print) IS - 1540-7748 (Electronic) IS - 0022-1295 (Linking) VI - 119 IP - 3 DP - 2002 Mar TI - Fast and slow voltage sensor movements in HERG potassium channels. PG - 275-93 AB - HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH(2)-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals. FAU - Smith, Paula L AU - Smith PL AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA. FAU - Yellen, Gary AU - Yellen G LA - eng GR - HL 57383/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Cation Transport Proteins) RN - 0 (Ether-A-Go-Go Potassium Channels) RN - 0 (KCNH6 protein, human) RN - 0 (Potassium Channels) RN - 0 (Potassium Channels, Voltage-Gated) SB - IM MH - Amino Acid Sequence MH - Animals MH - *Cation Transport Proteins MH - Ether-A-Go-Go Potassium Channels MH - Ion Channel Gating/drug effects/genetics/*physiology MH - Molecular Sequence Data MH - Mutation/genetics/physiology MH - Potassium Channels/genetics/*physiology MH - *Potassium Channels, Voltage-Gated MH - Xenopus PMC - PMC2217288 EDAT- 2002/02/28 10:00 MHDA- 2002/06/25 10:01 CRDT- 2002/02/28 10:00 PHST- 2002/02/28 10:00 [pubmed] PHST- 2002/06/25 10:01 [medline] PHST- 2002/02/28 10:00 [entrez] AID - 8534 [pii] AID - 10.1085/jgp.20028534 [doi] PST - ppublish SO - J Gen Physiol. 2002 Mar;119(3):275-93. doi: 10.1085/jgp.20028534. PMID- 11773240 OWN - NLM STAT- MEDLINE DCOM- 20020319 LR - 20190508 IS - 0022-1295 (Print) IS - 1540-7748 (Electronic) IS - 0022-1295 (Linking) VI - 119 IP - 1 DP - 2002 Jan TI - Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel. PG - 83-91 AB - Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Our previous work using the specific HCN channel blocker ZD7288 provided evidence for an intracellular activation gate for these channels because it appears that ZD7288, applied from the intracellular side, can enter and leave HCN channels only at voltages where the activation gate is opened (Shin, K.S., B.S. Rothberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101). However, the ZD7288 molecule is larger than the Na(+) or K(+) ions that flow through the open channel. In the present study, we sought to resolve whether the voltage gate at the intracellular entrance to the pore for ZD7288 also can be a gate for permeant ions in HCN channels. Single residues in the putative pore-lining S6 region of an HCN channel (cloned from sea urchin; spHCN) were substituted with cysteines, and the mutants were probed with Cd(2+) applied to the intracellular side of the channel. One mutant, T464C, displayed rapid irreversible block when Cd(2+) was applied to opened channels, with an apparent blocking rate of approximately 3 x 10(5) M(-1)s(-1). The blocking rate was decreased for channels held at more depolarized voltages that close the channels, which is consistent with the Cd(2+) access to this residue being gated from the intracellular side of the channel. 464C channels could be recovered from Cd(2+) inhibition in the presence of a dithiol applied to the intracellular side. The rate of this recovery also was reduced when channels were held at depolarized voltages. Finally, Cd(2+) could be trapped inside channels that were composed of WT/464C tandem-linked subunits, which could otherwise recover spontaneously from Cd(2+) inhibition. Thus, Cd(2+) escape is also gated at the intracellular side of the channel. Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore. FAU - Rothberg, Brad S AU - Rothberg BS AD - Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA. FAU - Shin, Ki Soon AU - Shin KS FAU - Phale, Prashant S AU - Phale PS FAU - Yellen, Gary AU - Yellen G LA - eng GR - HL 57383/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Cyclic Nucleotide-Gated Cation Channels) RN - 0 (HCN1 protein, human) RN - 0 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels) RN - 0 (Ion Channels) RN - 0 (Nerve Tissue Proteins) RN - 0 (Potassium Channels) RN - J6K4F9V3BA (Cadmium Chloride) RN - K848JZ4886 (Cysteine) SB - IM MH - Animals MH - Binding Sites/genetics MH - Cadmium Chloride/pharmacology MH - Cell Line MH - Cyclic Nucleotide-Gated Cation Channels MH - Cysteine/genetics MH - Humans MH - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels MH - Intracellular Fluid/drug effects/*physiology MH - Ion Channel Gating/genetics/*physiology MH - Ion Channels/genetics/*physiology MH - Membrane Potentials/drug effects/genetics/physiology MH - Mutagenesis, Site-Directed MH - *Nerve Tissue Proteins MH - Point Mutation MH - Potassium Channels MH - Sea Urchins PMC - PMC2233860 EDAT- 2002/01/05 10:00 MHDA- 2002/03/20 10:01 CRDT- 2002/01/05 10:00 PHST- 2002/01/05 10:00 [pubmed] PHST- 2002/03/20 10:01 [medline] PHST- 2002/01/05 10:00 [entrez] AID - 8523 [pii] AID - 10.1085/jgp.119.1.83 [doi] PST - ppublish SO - J Gen Physiol. 2002 Jan;119(1):83-91. doi: 10.1085/jgp.119.1.83.