Career Advancement Award: Epithelial Chloride Channels

氯离子通道在上皮细胞离子转运和细胞体积调节中起关键作用。 然而，对氯离子通道功能和调节的分子机制知之甚少。 培养的两栖动物肾细胞系A6是一个独特的适合这种研究的模型，因为这些细胞分化良好，并具有强大的，神经调节，钠和氯离子转运机制。 目前，我们缺乏足够的知识的结构负责门控上皮氯离子通道，其膜定位，和调节通道活性的激素和细胞内信使的机制。 Wills博士将使用PCR和表达克隆方法从A6上皮中分离、测序和功能性表达氯离子通道。 在初步研究中，她已经表明，类似于ClC家族和pICln的氯离子通道存在于这些细胞中。 Wills博士将获得这些蛋白质的全长克隆，并在非洲爪蟾卵母细胞或昆虫（SF 9）细胞中表达后，通过全细胞电流或单通道测量测定其通道特性。 这些研究的结果将有助于上皮氯离子通道功能的基础知识，并将允许两个新的ClC氯离子通道的表征。 A6两栖动物肾上皮细胞的独特功能，应有利于这些氯离子通道的功能表达，并允许首次表达的野生型克隆的approximately调节ClC通道。 该项目将为未来上皮细胞离子通道的结构-功能研究奠定基础。

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.