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The Journal of Neuroscience, October 14, 2009, 29(41):13063-13073; doi:10.1523/JNEUROSCI.3193-09.2009

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Cellular/Molecular
Bestrophin-1 Encodes for the Ca²⁺-Activated Anion Channel in Hippocampal Astrocytes

Hyungju Park,^1 * Soo-Jin Oh,^1 * Kyung-Seok Han,^1,4 Dong Ho Woo,^1,4 Hyekyung Park,¹ Guido Mannaioni,² Stephen F. Traynelis,³ and C. Justin Lee^1,4

¹Center for Neural Science, Convergence Technology Laboratory, Korea Institute of Science and Technology, Seoul 136-791, Korea, ²Department of Pharmacology, University of Florence, 50121 Florence, Italy, ³Department of Pharmacology, Emory University, Atlanta, Georgia 30322, and ⁴Neuroscience Program, University of Science and Technology, Daejeon 305-701, Korea

Correspondence should be addressed to Dr. C. Justin Lee, Center for Neural Science, Convergence Technology Laboratory, Korea Institute of Science and Technology, 39-1 Hawolgok-Dong, Seongbuk-Gu, Seoul 136-791, Korea. Email: cjl{at}kist.re.kr

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_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.

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Received July 3, 2009; revised Sept. 7, 2009; accepted Sept. 9, 2009.

Correspondence should be addressed to Dr. C. Justin Lee, Center for Neural Science, Convergence Technology Laboratory, Korea Institute of Science and Technology, 39-1 Hawolgok-Dong, Seongbuk-Gu, Seoul 136-791, Korea. Email: cjl{at}kist.re.kr

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