Career Advancement Award: Epithelial Chloride Channels

氯通道在细胞内上皮离子转运和细胞体积调节中起着关键作用。然而，对氯通道的作用和调控的分子机制却知之甚少。培养的两栖类肾脏细胞系A6是唯一适合于此类研究的模型，因为这些细胞分化良好，具有强大的、受激素调节的钠和氯运输机制。目前，我们对上皮氯离子通道的门控结构、膜的定位以及激素和细胞内信使调节通道活性的机制缺乏足够的了解。威尔斯博士将使用聚合酶链式反应和表达克隆方法从A6上皮分离、测序和功能表达氯离子通道。在初步研究中，她已经证明，在这些细胞中存在类似于ClC家族和pICln的氯通道。Wills博士将获得这些蛋白质的全长克隆，并在非洲爪哇卵母细胞或昆虫(SF9)细胞中表达后，通过全细胞电流或单通道测量来分析它们的通道特性。这些研究的结果将有助于对上皮氯离子通道功能的基本了解，并将有助于表征两种新的CLC氯离子通道。A6两栖类肾上皮细胞的独特特性应该有助于这些氯离子通道的功能表达，并允许首次表达渗透调节的CLC通道的野生型克隆。该项目将为进一步研究上皮细胞离子通道的结构和功能奠定基础。

Chloride channels play key roles in epithelial ion transport in cells and in regulation of cell volume. However, little is known about the molecular mechanisms of chloride-channel function and regulation. The cultured amphibian renal-cell line A6 is a uniquely suited model for such studies because these cells are well differentiated and have robust, hormonally regulated, sodium and chloride transport mechanisms. At present we lack sufficient knowledge about the structures responsible for gating of epithelial chloride channels, their membrane localization, and the mechanisms for modulation of channel activity by hormones and intracellular messengers. Dr. Wills will use PCR and expression-cloning methods to isolate, sequence, and functionally express chloride channels from A6 epithelium. In preliminary studies, she has shown that chloride channels similar to the ClC family and pICln are present in these cells. Dr. Wills will obtain full-length clones for these proteins and assay their channel properties from whole-cell current or single-channel measurements following expression in Xenopus oocytes or in insect (SF9) cells. The results of these studies will contribute to basic knowledge of epithelial chloride-channel function and will allow characterization of two novel ClC chloride channels. The unique features of A6 amphibian renal-epithelial cells should facilitate the functional expression of these chloride channels and allow the first expression of wild-type clones of osmotically-regulated ClC channels. The project will provide the foundation for future structure-function studies of epithelial ion channels.