CALCIUM SITES IN CALCIUM CHANNEL GATING

电压(V)操作的钙通道是信号转导的关键，并且 参与许多基本职能。CA渠道(或密切相关的 骨骼肌分子类)具有附加功能， 用于激励-收缩耦合的电压传感。这种二元性告诉我们 本提案中，关于钙通道门控的知识是 应用于骨骼肌电压传感器，并且相反地，选择 对电压传感器的观测结果进行了外推，以扩展我们的 对钙通道门控的理解。 门控的三个方面将在心脏(CARD)和 骨骼(SKM)钙通道：电压依赖性失活、钙依赖性 失活和钙介导的正反馈。有一种钙结合 影响这些功能的站点，可能位于 通道α1亚基。这些地点的位置和性质将是 探索过了。首先，每个站点都将在 异源表达“野生型”的原生系统和哺乳动物系统 通道蛋白。随后，这些通道将被突变修改，并且 功能后果将在表达系统中进行评估。 在心脏L的经络中，我们将探索其属性 Site“，防止SKM和Card失活的钙结合位点 通道，然后检验假设I，即启动部位涉及 决定渗透的离子选择性的相同区域。假设II 是钙结合到α1亚单位的细胞质区域并导致 他们关闭了航道。选定的区域将在以下区域之间交换 心脏和骨骼α1亚单位，或突变以破坏钙结合 性格。假说三是钙结合到一个细胞质区域。 Alpha1增强激活。两个区域将突变为 降低或提高它们的钙结合能力。解释…的差异 哺乳动物和两栖动物的SKM通道之间的正反馈， 林蛙心肌和骨骼肌钙通道的主要亚基 卡特斯贝纳将被克隆。然后，这些两栖克隆人将用于 类似于哺乳动物克隆的表达实验。 拟议中的工作以及其他实验室的工作结果将是 根据不断发展的渠道门控模型进行解释，这已经 在内口结合了离子扩散方面，应该给出 当这项工作完成时，对分子方面的描述要详细得多。 目前的提案对于增进了解也将是重要的 肌肉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.