Qu Zhiqiang, Wei Raymond W, Mann Wesley, Hartzell H Criss
Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2003 Dec 5;278(49):49563-72. doi: 10.1074/jbc.M308414200. Epub 2003 Aug 25.
Ca2+-activated Cl- channels play important diverse roles from fast block to polyspermy to olfactory transduction, but their molecular identity has not been firmly established. By searching sequence databases with the M2 pore domain of ligand-gated anion channels, we identified potential Ca2+-activated Cl- channels, which included members of the bestrophin family. We cloned two bestrophins from Xenopus oocytes, which express high levels of Ca2+-activated Cl- channels. The Xenopus bestrophins were expressed in a variety of tissues. We predict that bestrophin has six transmembrane domains with the conserved RFP domain playing an integral part in ionic selectivity. When Xenopus bestrophins were heterologously expressed in human embryonic kidney-293 cells, large Ca2+-activated Cl- currents were observed. The currents are voltage- and time-independent, do not rectify, have a Kd for Ca2+ of approximately 210 nm, and exhibit a permeability ratio of I- > Br- > Cl- >> aspartate. The W93C and G299E mutations produce non-functional channels that exert a dominant negative effect on wild type channels. We conclude that bestrophins are the first molecularly identified Cl- channels that are dependent on intracellular Ca2+ in a physiological range.
钙离子激活的氯离子通道发挥着重要且多样的作用，从快速阻断多精入卵到嗅觉转导，但它们的分子身份尚未完全确定。通过利用配体门控阴离子通道的M2孔道结构域搜索序列数据库，我们鉴定出了潜在的钙离子激活的氯离子通道，其中包括贝斯特罗芬家族成员。我们从非洲爪蟾卵母细胞中克隆了两种贝斯特罗芬，这些卵母细胞表达高水平的钙离子激活的氯离子通道。非洲爪蟾的贝斯特罗芬在多种组织中表达。我们预测贝斯特罗芬有六个跨膜结构域，保守的RFP结构域在离子选择性中起重要作用。当非洲爪蟾的贝斯特罗芬在人胚肾-293细胞中异源表达时，观察到了大的钙离子激活的氯离子电流。这些电流与电压和时间无关，不发生整流，钙离子的解离常数约为210纳米，并且表现出碘离子＞溴离子＞氯离子＞＞天冬氨酸的通透率。W93C和G299E突变产生无功能的通道，对野生型通道产生显性负效应。我们得出结论，贝斯特罗芬是首个在分子水平上鉴定出的、在生理范围内依赖细胞内钙离子的氯离子通道。
