Mechanisms of Ca2+ and voltage-dependent inactivation Ca channels

描述(申请人提供)：高压激活的钙通道(CaV1/2通道)传递钙内流，驱动一系列生物功能，包括心脏兴奋、神经传递和记忆形成。因此，它们受到钙依赖(CDI)和电压依赖失活(VDI)的严格调控。虽然这些过程的初始步骤是众所周知的，但对后续步骤却知之甚少。这些未知数是该领域的显著空白，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。该方案的总体目标是确定去极化或钙与CaM结合后的中间事件，并通过三个目标来阐明CDI和VDI的最终构象：(1)钙调蛋白(Calvi)是否通过在不同的通道结构域之间移动来诱导CDI？初步数据表明，通道C末端上游EF-Hand结构域的突变可以显著降低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EF-Hand基序在决定CDI中的结构和功能作用，(B)探索CaM是否离开其已知的IQ结合部位，以及(C)在膜片钳下使用TIRF/FRET成像监测由此产生的细胞内结构域的构象变化。(2)机械扭转细胞内环是否导致钙通道失活？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FKBP通道环和膜定位的Lyn-FRB结构域之间的异二聚化来诱导调用这些约束。(3)CDI和VDI的最终阶段流程是什么？CDI和VDI的终末期机制的三种主要模型是：(I)铰链盖闭塞，(Ii)孔洞塌陷，或(病态)变构抑制开放。初步数据表明，CDI是通过变构调节发生的。相关的孔洞塌陷的分子机制可能在钙通道中是保守的。我们将通过以下方式区分这些机制：(A)共表达L-11环肽，这可能作为加速失活的游离‘盖’的过剩；(B)进行选择性滤膜区域的丙氨酸扫描以测试孔洞塌陷；以及(C)使用FRET来成像与通道失活相关的可能的孔洞塌陷。相关性：通过这些实验，我们将填补目前对钙通道失活以及更广泛地说，离子通道调节的理解中的一个很大空白。它还将提供一个垫脚石，促进我们对致命性通道病的机制后果的理解，为有针对性的治疗干预带来希望。

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.