Bestrophin-1 Encodes for the Ca2+ Activated Anion Channel in hippocampal astrocytes

Bestrophin-1 Encodes for the Ca2+ Activated Anion Channel in hippocampal astrocytes
오수진박형주한경석우동호Guido MannaioniStephen F. Traynelis이창준
Astrocyte; Ca2+-activated anion channel (CAAC); Bestrophin; protease-activated receptor-1(PAR-1); Gq-coupled receptor; short-hairpin RNA (shRNA)
Issue Date
International Society for Neurochemistry
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 produced inward current at holding potential of -70 mV that was dependent on increase in intracellular Ca2+ transients upon Gαq-coupled receptor activation. This current was mediated mostly by anions and sensitive to well-known anion channel blockers such as niflumic acid, NPPB, and flufenamic acid. To find the molecular identity of an anion channel responsible for the CAAC current, we analyzed the expression of candidate genes and found that mRNA for mouse Bestrophin 1 (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 shRNA, demonstrating that the CAAC is mediated by a channel encoded by mBest1. Finally, hippocampal CA1 astrocytes also showed mBest1-mediated CAAC as tested mBest1-specific shRNA, proposing that mBest1 encodes CAAC in astrocytes in situ. Collectively, our data provide molecular evidence that mBest1 channel is responsible for CAAC function in astrocytes.
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