Calcium-dependent gating of the L-type Calcium Channel

描述(申请人提供)：电压门控L型钙通道的钙依赖失活在心脏生理学中具有重要意义。它是动作电位时程的主要决定因素，因此是兴奋-收缩偶联的重要调节因子。在心力衰竭的干扰下，CDI异常可能是心律失常发生的触发因素。CDI也是反馈抑制的一个突出例子，在这种情况下，渗透到通道的钙离子本身就会触发通道加速失活。本建议旨在通过对最近发现的分子成分的研究来确定CDI的分子机制：1)钙传感器是钙调素(CaM)，结构性地与通道的成孔亚单位α1c结合；2)α1c中的CDI效应器是“IQ基序”，即α1c C末端的CaM结合部位；目的1)确定CaM在失活调控中的结构作用；2)确定IQ基序在失活调控中的结构作用；3)确定EF-Hand(S)在CDI中的作用。前两个目标将确定CaM和IQ基序的分子作用，这两个基序在钙依赖和非钙非依赖性失活中都是必不可少的。第三个目标就有争议的EF-Hand基序在阿尔法1c中的作用提出了新的假设。我们已经鉴定了第二个EF-手，并显示了初步的结果表明，这两个EF-手形成了一对结构对，类似于CaM的单个叶。我们的初步数据允许我们提出一个模型，该模型结合了CaM、IQ基序和EF-Hand对在调节失活中的作用。这些新的假设有望显著改变目前的CDI模型，也为理解电压门控通道失活提供了一个模板。这项工作将加深我们对正常心脏功能以及这些过程如何在心力衰竭和心律失常等病理生理状态下受到干扰的理解。

DESCRIPTION (provided by applicant): Calcium-dependent inactivation (CDI) of voltage-gated L-type Ca2+ channels is of fundamental importance in cardiac physiology. It is a major determinant of the action potential duration and thus an essential regulator of excitation-contraction coupling. Disturbed in heart failure, abnormal CDI may be a trigger for arrhythmogenesis. CDI is also a pre-eminent example of feedback inhibition, in which the very Ca2+ that permeates the channel triggers accelerated channel inactivation. This proposal aims to identify the molecular mechanisms underlying CDI by focusing on the molecular components that have recently been identified: 1) the Ca2-sensor is calmodulin (CaM), constitutively bound to the channel's pore-forming subunit alpha 1c; 2) the CDI effector in alpha 1c is the "IQ motif', a CaM binding site in the alpha 1c C-terminal tail; and 3) an EF-hand motif in the C-terminal tail serves an essential structural role.This proposal contains three specific aims: Aim 1) Determine the structural role of CaM in the regulation of inactivation Aim 2) Determine the structural role of the IQ motif in the regulation of inactivation and Aim 3) Define the role of the EF-hand(s) in CDI. The first two Aims will identify the molecular roles of CaM and the IQ motif, essential in both Ca2+-dependent and Ca2+-independent inactivation. The third Aim proposes novel hypotheses regarding the role of the controversial EF-hand motif within alpha 1c. We have identified a second EF-hand, and show preliminary results indicating that the two EF-hands form a structural pair, similar to a single lobe of CaM. Our preliminary data allow us to propose a model that incorporates how CaM, the IQ motif, and the EF-hand pair function in regulating inactivation. These novel hypotheses promise to significantly alter the current model of CDI and also provide a template for the understanding of voltage-gated channel inactivation. This work will augment our understanding of normal cardiac function and how these processes are perturbed under pathophysiological states, such as heart failure and arrhythmogenesis.