Ion Channel Regulation by Ca2+

Ca2 的离子通道调节

• 批准号: 7090004
• 负责人: Christopher J Lingle
• 金额: $ 26.59万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7090004
• 关键词: Xenopus; Xenopus oocyte; calcium; calcium channel; intermolecular interaction; membrane channels; molecular site; potassium channel; protein structure function; protein transport; voltage gated channel

DESCRIPTION (provided by applicant): This project will examine the mechanisms and sites of action of divalent cations that regulate the behavior of calcium (Ca2+)- and voltage-activated BK-type Slo1 potassium channels. The goal of the experiments is to define the multiple sites at which Ca2+ and Mg2+ act on Slo1 channels and the mechanism by which these sites may regulate channel function. These goals will be met through the concerted use of biophysical, biochemical, and molecular biological approaches. Previous work has shown that Slo1 channel function is regulated by at least two divalent cation sites with differing affinities and a powerful mechanistic framework for analyzing these allosteric effects has been developed. To define the locations and functional roles of these sites, the homologous Slo3 variant which lacks the key divalent cation regulatory sites will be employed. Chimeric constructs of Slo1/Slo3 subunits will be examined to determine regions that confer low and high affinity regulation by Ca2+ and Mg2+. These results will be analyzed in terms of allosteric models that allow definition of the presence of particular binding sites and the affinity of those sites for divalent cations. Complementary biochemical work will also be done on fusion peptides obtained from portions of the Slo1 subunit to demonstrate whether particular regions may specifically bind Ca2+ and/or Mg2+. The proposed work builds on previous work from this lab, and is expected to lead to insights into the fundamental mechanisms by which binding of Ca2+ and other divalent cations to a protein can regulate the functional properties of that protein. Because of the ubiquitous importance of Ca2+ as an intracellular messenger, understanding how Ca2+ concentrations spanning over four orders of magnitude can regulate the function of the Slo1 channel protein is of importance to the general question of how Ca2+-sensing proteins detect and respond to Ca2+.

描述（由申请人提供）：本项目将研究调节钙（Ca 2+）和电压激活的BK型Slo 1钾通道行为的二价阳离子的作用机制和作用部位。实验的目的是确定多个网站在其中Ca 2+和Mg 2+作用于Slo 1通道和这些网站可以调节通道功能的机制。这些目标将通过协调一致地使用生物物理、生物化学和分子生物学方法来实现。以前的工作表明，Slo 1通道的功能是由至少两个二价阳离子位点与不同的亲和力和一个强大的机制框架，用于分析这些变构效应已被开发。为了确定这些位点的位置和功能作用，将采用缺乏关键二价阳离子调控位点的同源Slo 3变体。将检查Slo 1/Slo 3亚基的嵌合构建体，以确定通过Ca 2+和Mg 2+赋予低和高亲和力调节的区域。这些结果将分析变构模型，允许定义的特定结合位点的存在和这些网站的二价阳离子的亲和力。还将对从Slo 1亚基部分获得的融合肽进行补充生物化学工作，以证明特定区域是否可以特异性结合Ca 2+和/或Mg 2+。 拟议的工作建立在该实验室以前的工作基础上，预计将导致对Ca 2+和其他二价阳离子与蛋白质结合的基本机制的深入了解，可以调节该蛋白质的功能特性。由于Ca 2+作为细胞内信使的普遍重要性，了解跨越四个数量级的Ca 2+浓度如何调节Slo 1通道蛋白的功能对于Ca 2+敏感蛋白如何检测和响应Ca 2+的一般问题具有重要意义。