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: 8288298
• 负责人: Manu Ben Johny
• 金额: $ 4.22万
• 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/8288298
• 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）传达了钙涌入，这些钙涌入可驱动大量生物学功能，包括心脏激发，神经传递和记忆形成。因此，它们受钙依赖性（CDI）和依赖性灭活（VDI）的严格调节。尽管这些过程的初始步骤是众所周知的，但对后续步骤知之甚少。这些未知数是该领域的显着差距，因为这种失活的缺陷会导致各种神经元和心脏钙通道病。该提案的总体目标是确定遵循去极化或CA与CAM结合的中间事件，并通过3个目标阐明CDI和VDI的最终构象：（1）Calmodulin（CalVI）是否通过在不同的通道域之间移动CDI？ 初步数据表明，通道的C末端上游EF手中的突变可能会大大减少CDI。我们还在计算上确定了潜在的替代凸轮结合位点。我们将（a）确定EF手基序的结构和功能作用在确定CDI时至关重要的（b）探索CAM是否留下其众所周知的智商结合位点，以及（c）使用TIRF/FRET成像在斑块夹下方监测细胞内结构域的构象变化。 （2）细胞内环上的机械扭转会诱导Ca2+通道灭活吗？如果S6门与通道上的细胞内环的机械耦合在灭活中起作用，则我们应该能够通过物理限制这些回路来改变通道失活。我们将通过雷帕霉素诱导的FKBP渠道环与膜 - 位置的LYN-FRB结构域之间的异二聚化引起诱导的限制。 （3）CDI和VDI的最终终点过程是什么？ CDI和VDI终阶段机制的三个主要模型是：（i）铰链嵌入式遮挡，（ii）孔隙折叠或（不良）对开放的变构抑制。初步数据表明，CDI通过变构调节发生。孔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）共表达的L-LL环路肽可以充当过度灭活的自由“盖子”，（b）进行选择性滤波器区域的丙氨酸扫描，以测试孔塌陷，并使用FRET使用FRET来形象可能与通道灭活相关的孔隙率。 相关性：通过这些实验，我们将在当前对CA通道失活的理解和更一般而言的离子通道调节中填补一个大空白。这也将提供一块垫脚石，以促进我们对致命通道病的机理后果的理解，从而提高了对有针对性的治疗干预措施的希望。