Mechanisms of Ca2+ and voltage-dependent inactivation Ca channels

描述（由申请人提供）：高压激活的钙通道（CaV1/2通道）传递钙内流，驱动大量的生物功能，包括心脏兴奋、神经传递和记忆形成。因此，它们受到钙依赖性（CDI）和电压依赖性失活（VDI）的严格调节。虽然这些过程的最初步骤是众所周知的，但对后续步骤知之甚少。这些未知是该领域的突出空白，因为这种失活的缺陷会导致各种神经元和心脏钙通道病变。本提案的总体目标是确定去极化或Ca与CaM结合后的中间事件，并通过3个目标阐明CDI和VDI的最终构象：(1)钙调素（Calmodulin, CalVI）是否通过在不同通道域之间移动来诱导CDI ？初步数据表明，通道c端上游EF-hand结构域的突变可显著降低CDI。我们还通过计算确定了潜在的替代CaM结合位点。我们将(a)确定在决定CDI中至关重要的EF-hand基模的结构和功能作用，(b)探索CaM是否离开其众所周知的IQ结合位点，以及(c)在膜片钳下使用TIRF/FRET成像监测细胞内结构域的构象变化。(2)细胞内环的机械扭转是否诱导Ca2+通道失活？如果S6门与通道上的细胞内环的机械耦合在失活中起作用，我们应该能够通过物理限制这些环来改变通道失活。我们将通过雷帕霉素诱导的fkbp通道环和膜定位的Lyn-FRB结构域之间的异二聚化诱导地调用这种约束。(3) CDI和VDI的最终终末过程是什么？CDI和VDI终末期机制的三种主要模型是：(i)铰盖闭塞，（ii）孔塌陷，或（ii）开口变构抑制。初步资料表明，CDI是通过变构调节发生的。孔隙崩塌的相关分子机制可能在钙通道中保守。我们将通过以下方式来区分这些机制：(a)共同表达l-ll环肽，它可以作为加速失活的过量自由“盖子”，(b)对选择性过滤区域进行丙氨酸扫描以测试孔塌陷，以及(c)使用FRET成像可能与通道失活相关的孔塌陷。相关性：通过这些实验，我们将填补我们目前对Ca通道失活和更普遍的离子通道调节的理解的巨大空白。它也将提供一个垫脚石，以促进我们对致命通道病变的机制后果的理解，提高对靶向治疗干预的希望。

DESCRIPTION (provided by applicant): High-voltage activated Ca channels (CaV1/2 channels) convey calcium influx that drives a vast array of biological functions, including cardiac excitation, neurotransmission, and memory formation. As such, they are tightly regulated by calcium-dependent (CDI) and voltage dependent inactivation (VDI). While the initial steps of these processes are well-known, little is known about the subsequent steps. These unknowns are prominent gaps in the field, given that defects in such inactivation cause various neuronal and cardiac calcium channelopathies. The overall goal of this proposal is to identify the intermediate events that follow depolarization or Ca binding to CaM and to elucidate the final conformations of CDI and VDI through 3 aims: (1) Does Calmodulin(CalVI) induce CDI by moving among different channel domains? Preliminary data indicate that mutations in an upstream EF-hand domain on C-terminus of channel could dramatically reduce CDI. We have also identified potential alternate CaM binding sites computationally. We will (a) identify the structural and functional roles of the EF-hand motif critical in determining CDI, (b) explore whether CaM leaves its well-known IQ binding site, and (c) monitor resulting conformation changes of intracellular domains using TIRF/FRET imaging under patch clamp. (2) Does mechanical torsion on intracellular loops induce Ca2+ channel inactivation? If mechanical coupling of the S6 gates with the intracellular loops on the channel play a role in inactivation, we should be able to alter channel inactivation by physically constraining these loops. We will inducibly invoke such constraints by rapamycin-induced heterodimerization between FKBP-channel loops and membrane-localized Lyn-FRB domains. (3) What are the ultimate end-stage processes of CDI and VDI? Three major models for end-stage mechanisms of CDI and VDI are: (i) hinged-lid occlusion, (ii) pore-collapse, or (ill) allosteric inhibition of opening. Preliminary data suggests that CDI occurs through allosteric modulation. The relevant molecular machinery for pore collapse is possibly conserved in Ca channels. We will distinguish among these mechanisms by: (a) co-expressing l-ll loop peptide which could act as an excess of free 'lids' that speed inactivation, (b) undertaking an alanine scan of selectivity filter regions to test for pore collapse, and (c) using FRET to image possible pore-collapse associated with channel inactivation. Relevance: Through these experiments we would fill a large void in our present understanding of Ca channel inactivation and, more generally, ion channel regulation. It would also provide a stepping stone to advance our understanding of the mechanistic consequences of lethal channelopathies raising hope for targeted therapeutic interventions.