Regulation of Calcium Activated Chloride Channels

描述(申请人提供)：肾脏的功能是维持血液中正常的离子浓度。正常情况下，肾脏将血液离子成分维持在非常狭窄的范围内，不平衡会导致非常严重的健康后果，包括死亡。这项资助的目标是了解肾脏中起作用的离子通道，以运输氯离子并维持体内氯离子的动态平衡。这个应用程序将专注于刚刚发现的一个令人兴奋的新的氯离子通道家族，anocTamins(也称为TMEM16)。我们已有初步数据表明，肾脏中表达了几种异构体蛋白，它们可能在氯的分泌和/或吸收中发挥重要作用。这项应用将研究Anotamins在正常小鼠和人类肾脏以及人类和多囊肾病小鼠模型肾脏中的表达。将使用定量RT-PCR、Western印迹和免疫荧光共聚焦显微镜来确定其表达。使用电生理学(全细胞和单通道膜片钳记录)和表达Ano1的细胞的生化分析来研究Anoctamin通道的功能结构和组织。我们将探索钙结合部位的位置以及电压和钙离子对Ano1的调节机制，以开发一个定量的模型来研究Anoctaamin通道是如何被门控的。

DESCRIPTION (provided by applicant): The function of the kidneys is to maintain normal ionic concentrations in the blood. Normally the kidneys maintain the blood ionic composition within very tight range and imbalance produces very serious health consequences including death. The goal of this grant is to understand the ion channels that function in the kidney to transport chloride and maintain chloride homeostasis in the body. This application will focus on an exciting new family of chloride channels that was just discovered, the anoctamins (also called TMEM16). We have preliminary data that several anoctamins are expressed in kidney and are likely to play important roles in chloride secretion and/or absorption. This application will investigate the expression of anotamins in normal mouse and human kidney and in kidney from human and from mouse models of polycystic kidney disease. Expression will be determined using quantitative RT-PCR, western blot, and immunofluorescent confocal microscopy. The functional structure and organization of the anoctamin channel will be investigated using electrophysiological (whole-cell and single channel patch clamp recording) and biochemical analysis of cells expressing Ano1. The location of Ca2+ binding sites and mechanisms of regulation of Ano1 by voltage and Ca2+ will be explored to develop a quantitative model of how anoctamin channels are gated.