Molecular Cloning of Epithelial K Channels

上皮 K 通道的分子克隆

• 批准号: 7022319
• 负责人: HENRY SACKIN
• 金额: $ 35.8万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7022319
• 关键词: X ray crystallography; Xenopus; Xenopus oocyte; acidity /alkalinity; epithelium; kidney cell; molecular cloning; potassium channel; protein structure function; receptor; single cell analysis; voltage /patch clamp; voltage gated channel

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.

描述（由申请人提供）：拟议项目延续了最初的特定目标，即了解通过肾脏内向整流钾通道：ROMK（Kir1.1）的钾渗透和门控。然而，该项目现在包含了一个更一般的主题，即理解K通道门控的一般机制。鉴于最近对细菌KcsA和Ca激活的BK通道的晶体学研究表明K通道门控与特定的结构变化相关，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之间进行选择，而且还用作通道的外部门。与外门串联的第二个内门被认为是由内部跨膜螺旋的铰链部分组成。耦合到该内门的特定传感器（电压、配体、pH）将限定特定通道的功能特性。电生理学实验将进行测试这两个门的假设，使用ROMK及其突变体和嵌合体，在Zenopus卵母细胞中表达。卵母细胞的研究方法有：双电极电压钳（TEVC）、卵母细胞切开技术和单通道膜片钳记录。这个建议是一个独特的机会，联合收割机可用的晶体学信息与功能电生理学研究，以阐明可能是一个一般的范例K通道门控，以及一个特定的机制，调节肾脏K分泌。