title: Bestrophin-1 encodes for the Ca2+-activated anion channel in hippocampal astrocytes.
abstract: In mammalian brain, neurons and astrocytes are reported to express various chloride and anion channels, but the evidence for functional expression of Ca(2+)-activated anion channel (CAAC) and its molecular identity have been lacking. Here we report electrophysiological evidence for the CAAC expression and its molecular identity by mouse Bestrophin 1 (mBest1) in astrocytes of the mouse brain. Using Ca(2+) imaging and perforated-patch-clamp analysis, we demonstrate that astrocytes displayed an inward current at holding potential of -70 mV that was dependent on an increase in intracellular Ca(2+) after G(alphaq)-coupled receptor activation. This current was mediated mostly by anions and was sensitive to well known anion channel blockers such as niflumic acid, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and flufenamic acid. To find the molecular identity of the anion channel responsible for the CAAC current, we analyzed the expression of candidate genes and found that the mRNA for mouse mBest1 is predominantly expressed in acutely dissociated or cultured astrocytes. Whole-cell patch-clamp analysis using HEK293T cells heterologously expressing full-length mBest1 showed a Ca(2+)-dependent current mediated by mBest1, with a complete impairment of the current by a putative pore mutation, W93C. Furthermore, mBest1-mediated CAAC from cultured astrocytes was significantly reduced by expression of mBest1-specific short hairpin RNA (shRNA), suggesting that the CAAC is mediated by a channel encoded by mBest1. Finally, hippocampal CA1 astrocytes in hippocampal slice also showed mBest1-mediated CAAC because it was inhibited by mBest1-specific shRNA. Collectively, these data provide molecular evidence that the mBest1 channel is responsible for CAAC function in astrocytes.
authors.affiliation: Center for Neural Science, Convergence Technology Laboratory, Korea Institute of Science and Technology, Seoul 136-791, Korea.
authors.forename: Hyungju
authors.initials: H
authors.lastname: Park
authors.forename: Soo-Jin
authors.initials: SJ
authors.lastname: Oh
authors.forename: Kyung-Seok
authors.initials: KS
authors.lastname: Han
authors.forename: Dong Ho
authors.initials: DH
authors.lastname: Woo
authors.forename: Hyekyung
authors.initials: H
authors.lastname: Park
authors.forename: Guido
authors.initials: G
authors.lastname: Mannaioni
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Traynelis
authors.forename: C Justin
authors.initials: CJ
authors.lastname: Lee
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000502:physiology*; D000075002:Bestrophins; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002614:Chelating Agents / Q000494:pharmacology; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D015232:Dinoprostone / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004558:Electric Stimulation / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005136:Eye Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D006624:Hippocampus / Q000166:cytology*; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels; D008246:Lysophospholipids / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009842:Oligopeptides / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011760:Pyrrolidinones / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D019284:Thapsigargin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection / Q000379:methods
chemical_list: C491856:Best1 protein, mouse; D000075002:Bestrophins; D002614:Chelating Agents; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D004963:Estrenes; D005136:Eye Proteins; D007473:Ion Channels; D008246:Lysophospholipids; D009842:Oligopeptides; C462999:PAR-1-activating peptide; D011760:Pyrrolidinones; D034741:RNA, Small Interfering; D013873:Thionucleotides; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D015232:Dinoprostone; C032881:lysophosphatidic acid; D001920:Bradykinin; D002118:Calcium; C008501:2-methylthio-ATP