Molecular Cloning of Epithelial K Channels

描述(申请人提供)：建议的项目延续了最初的具体目标，即了解钾通过肾脏的渗透和门控，向内整流，钾通道：ROMK(Kir1.1)。然而，该项目现在包含了一个更一般的主题，即了解K通道门控的一般机制。鉴于最近对细菌KCSA和钙激活的BK通道的结晶学研究表明K通道门控与特定的结构变化有关，这是特别及时的。在该模型中，K选择性过滤器不仅在阳离子中进行选择，而且还起到了沟道外栅的作用。与外门串联的第二个内门被认为是由内跨膜螺旋的铰链部分组成的。连接到该内部栅极的特定传感器(电压、配体、pH)将定义特定通道的功能特性。电生理实验将使用在泽诺普斯卵母细胞中表达的ROMK及其突变体和嵌合体来验证这一双门假说。卵母细胞的研究包括：双电极电压钳(TEVC)技术、卵母细胞切开技术和单通道膜片钳记录技术。这一建议是一个独特的机会，将现有的结晶学信息与功能电生理研究相结合，以阐明K通道门控的一般范例以及调节肾脏K分泌的特定机制。

DESCRIPTION (provided by applicant): The proposed project continues the original specific goal of understanding K permeation and gating through the renal, inward rectifying, K channel: ROMK (Kir1.1). However, the project now encompasses a more general theme of understanding the general mechanics of K channel gating. This is particularly timely in view of recent crystallographic studies on bacterial KcsA and Ca-activated BK channels suggesting that K channel gating is associated with specific structural changes. In this model, the K selectivity filter not only selects among cations but also functions as an outer gate for the channel. A second, inner gate, in series with the outer gate, is believed to consist of a hinged section of the inner transmembrane helix. Particular sensors (voltage, ligand, pH) that are coupled to this inner gate would define the functional characteristics of a particular channel. Electrophysiological experiments would be conducted to test this two-gate hypothesis, using ROMK and its mutants and chimeras, expressed in Zenopus oocytes. Oocytes would be studied with: the two electrode voltage clamp (TEVC), the cut-open oocyte technique and patch-clamp recording of single channels. This proposal is a unique opportunity to combine available crystallographic information with a functional electrophysiological study to elucidate what may be a general paradigm for K channel gating, as well as a specific mechanism for regulating renal K secretion.