Park, Hyungju Oh, Soo-Jin Han, Kyung-Seok Woo, Dong Ho Park, Hyekyung Mannaioni, Guido Traynelis, Stephen F. Lee, C. Justin 2024-01-20T20:32:11Z 2024-01-20T20:32:11Z 2021-09-04 2009-10-14 0270-6474 https://pubs.kist.re.kr/handle/201004/132035 In mammalian brain, neurons and astrocytes are reported to express various chloride and anion channels, but the evidence for functional expression of Ca2+-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 Ca2+ 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 Ca2+ after G(alpha q)-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 Ca2+-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. English SOC NEUROSCIENCE ACTIVATED CHLORIDE CURRENTS IN-SITU HYBRIDIZATION CL-CHANNEL MOUSE BESTROPHIN-2 CA2+ CHANNELS PROTEIN PERMEATION MEMBRANE TMEM16A FAMILY Bestrophin-1 Encodes for the Ca2+-Activated Anion Channel in Hippocampal Astrocytes Article 10.1523/JNEUROSCI.3193-09.2009 1 JOURNAL OF NEUROSCIENCE, v.29, no.41, pp.13063 - 13073 JOURNAL OF NEUROSCIENCE 29 41 13063 13073 scie scopus 000270845000040 2-s2.0-70350450278 Neurosciences Neurosciences & Neurology Article ACTIVATED CHLORIDE CURRENTS IN-SITU HYBRIDIZATION CL-CHANNEL MOUSE BESTROPHIN-2 CA2+ CHANNELS PROTEIN PERMEATION MEMBRANE TMEM16A FAMILY astrocyte Ca2+-activated anion channel (CAAC) bestrophin protease-activated receptor-1(PAR-1) Gq-coupled receptor short-hairpin RNA (shRNA)