Mechanisms of Ca2+ and voltage-dependent inactivation Ca channels

Ca2 和电压依赖性失活 Ca 通道的机制

• 批准号: 8106165
• 负责人: Manu Ben Johny
• 金额: $ 4.12万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8106165
• 关键词: Alanine; Binding; Binding Sites; Biological Process; C-terminal; Calcium; Calculi; Calmodulin; Cardiac; Complex; Coupling; Data; Defect; Distal; EF Hand Motifs; EF-Hand Domain; Elements; Event; Feedback; Fluorescence Resonance Energy Transfer; Goals; Heterodimerization; Homology Modeling; Image; Ion Channel; Mechanics; Membrane; Memory; Modeling; Molecular; Molecular Conformation; Monitor; Mutation; Neurons; Peptides; Play; Process; Proline; Regulation; Role; Scanning; Scheme; Signal Transduction; Sirolimus; Site; Speed; Staging; Structure; Tacrolimus Binding Proteins; Testing; Therapeutic Intervention; Torsion; insight; neurotransmission; patch clamp; prototype; research study; sensor; voltage

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.

描述（由申请人提供）：高压激活的 Ca 通道（CaV1/2 通道）输送钙离子流入，驱动多种生物功能，包括心脏兴奋、神经传递和记忆形成。因此，它们受到钙依赖性失活（CDI）和电压依赖性失活（VDI）的严格调节。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。鉴于这种失活的缺陷会导致各种神经元和心脏钙通道病，这些未知因素是该领域的显着差距。该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）钙调蛋白（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 末端上游 EF-hand 结构域的突变可以显着降低 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）确定对确定 CDI 至关重要的 EF-hand 基序的结构和功能作用，（b）探索 CaM 是否离开其众所周知的 IQ 结合位点，以及（c）使用 TIRF 监测细胞内结构域的构象变化/膜片钳下的FRET成像。 (2) 细胞内环的机械扭转是否会导致Ca2+通道失活？如果 S6 门与通道上细胞内环的机械耦合在失活中发挥作用，那么我们应该能够通过物理约束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来诱导调用此类约束。 （3）CDI和VDI的最终流程是什么？ CDI 和 VDI 终末期机制的三个主要模型是：(i) 铰接盖闭塞，(ii) 孔塌陷或（不适）开放的变构抑制。初步数据表明，CDI 通过变构调节发生。孔隙塌陷的相关分子机制可能在 Ca 通道中保守。我们将通过以下方式区分这些机制：(a) 共表达 l-ll 环肽，它可以充当过量的游离“盖子”，加速失活，(b) 对选择性过滤区域进行丙氨酸扫描以测试孔塌陷，(c) 使用 FRET 对与通道失活相关的可能的孔塌陷进行成像。 相关性：通过这些实验，我们将填补目前对 Ca 通道失活以及更普遍的离子通道调节的理解中的巨大空白。它还将为加深我们对致命离子通道病机制后果的理解提供一个垫脚石，为有针对性的治疗干预带来希望。