CALCIUM SITES IN CALCIUM CHANNEL GATING

电压（V）操纵的Ca通道对于信号转导至关重要， 参与许多重要的功能。钙通道（或密切相关的 骨骼肌的一类分子）具有额外的功能， 用于兴奋-收缩耦合的电压感测。这种二元性告诉我们 本建议，其中门控钙通道的知识， 施加到骨骼肌电压传感器，并且相反地，选择 对电压传感器的观察外推，以扩大我们的 了解钙通道门控。 门控的三个方面将在心脏（卡）和 骨骼肌（SkM）钙通道：电压依赖性失活，钙依赖性 失活和Ca介导的正反馈。存在Ca 2+结合 影响这些功能的一个部位，可能位于Ca 通道α 1亚基。这些网站的位置和性质将 探讨了首先，每个站点将在 天然系统和异源表达“野生型”的哺乳动物系统 通道蛋白之后，通道将通过突变进行修改， 将在表达系统中评估功能结果。 在心脏L通道中，我们将探索“启动”的性质， 位点”，防止SkM和Card失活的Ca结合位点 通道，然后检验假设I，即引发位点涉及 确定渗透的离子选择性的相同区域。假设二 钙离子与α 1亚基的细胞质区域结合， 关闭通道。选定的地区将在以下地区之间交换 心脏和骨骼α 1亚基，或突变破坏钙结合 性格假设III是Ca结合到细胞质区域， α 1增强激活。两个区域将突变为 降低或增加其钙结合能力。来解释 哺乳动物和两栖动物的SkM通道之间的正反馈， 蛙心肌和骨骼肌钙通道的主要亚基 Catesbeiana将被克隆。两栖动物克隆将被用于 表达实验类似于哺乳动物克隆。 来自所提议的工作以及其他实验室的工作的结果将在 根据不断发展的通道门控模型来解释， 在内口处包含离子扩散方面，并且应当给出 更详细的分子方面的描述。 本提案对促进理解也很重要， 肌肉的兴奋和收缩之间的联系

Voltage (V)-operated Ca channels are crucial for signal transduction, and participate in many essential functions. Ca channels (or a closely related class of molecules of skeletal muscle) have an additional function, voltage sensing for excitation-contraction coupling. This duality informs the present proposal, in which knowledge of gating of Ca channels is applied to the skeletal muscle voltage sensor, and, conversely, selected observations on the voltage sensor are extrapolated to expand our understanding of Ca channel gating. Three aspects of gating will be studied in both cardiac (Card) and skeletal (SkM) Ca channels: voltage-dependent inactivation, Ca-dependent inactivation and Ca-mediated positive feedback. There is a Ca2+-binding site that influences each of these functions, probably located on the Ca channel alpha1 subunit. The location and nature of these sites will be explored. First, each site will be characterized functionally in the native system and a mammalian system heterologously expressing "wild type" channel proteins. Later, the channels will be modified by mutations, and the functional consequences will be evaluated in the expression system. In the cardiac L channel, we will explore the properties of the "priming site", a Ca-binding site that prevents inactivation of the SkM and Card channels, and then test hypothesis I, that the priming site involves the same regions that determine ion selectivity of permeation. Hypothesis II is that Ca binds to cytoplasmic regions of the alpha1 subunit and causes them to close the channel. Selected regions will be exchanged between cardiac and skeletal alpha1 subunits, or mutated to destroy the Ca-binding character. Hypothesis III is that Ca binding to a cytoplasmic region of alpha1 potentiates activation. Two regions will be mutated to either reduce or increase their Ca-binding ability. To interpret differences in positive feedback between SkM channels from mammals and amphibians, the main subunit of the Ca channel in cardiac and skeletal muscle of Rana Catesbeiana will be cloned. The amphibian clones will then be used in expression experiments analogous to those with the mammalian clones. Results from the work proposed -and from work in other labs- will be interpreted in terms of an evolving model of channel gating, that already incorporates ion diffusional aspects at the inner mouth, and should give a much more detailed account of molecular aspects when this work is done. The present proposal will also be important to advance the understanding of the links between excitation and contraction in muscle.