Ion Channel Regulation by Ca2+

描述(由申请人提供)：本项目将研究调节钙(钙)和电压激活的BK型Slo1钾通道行为的二价阳离子的作用机制和作用部位。实验的目的是确定钙离子和镁离子作用于sL1通道的多个部位，以及这些部位可能调节通道功能的机制。这些目标将通过协调使用生物物理、生化和分子生物学方法来实现。前人的工作表明，slo1通道的功能受至少两个不同亲和力的二价阳离子位点的调节，并且已经建立了一个强大的机制框架来分析这些变构效应。为了确定这些位点的位置和功能作用，将使用缺乏关键二价阳离子调控位点的同源Slo3变体。将研究Slo1/Slo3亚基的嵌合结构，以确定钙离子和镁离子对低亲和力和高亲和力调节的区域。这些结果将根据变构模型进行分析，该模型允许定义特定结合部位的存在以及这些部位对二价阳离子的亲和力。还将对从slo1亚基部分获得的融合肽进行补充生化工作，以证明特定区域是否可能特定地与钙和/或镁结合。 这项拟议的工作建立在该实验室之前的工作基础上，预计将导致对钙离子和其他二价阳离子与蛋白质结合的基本机制的深入了解，通过这些机制可以调节蛋白质的功能特性。由于钙离子作为细胞内信使的重要性无处不在，因此了解跨越四个数量级的钙离子浓度如何调节slo1通道蛋白的功能，对于钙敏感蛋白如何检测和响应钙离子具有重要意义。

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+.