L-type Ca2+ Channel Regulation by Calmodulin and CaBP1

钙调蛋白和 CaBP1 对 L 型 Ca2 通道的调节

• 批准号: 10618394
• 负责人: JAMES B AMES
• 金额: $ 31.4万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618394
• 关键词: Actinin; Alzheimer' s Disease; Binding; Binding Sites; Biological Assay; Brain; C-terminal; CTF1 gene; Calcium; Calcium Binding; Calcium Channel; Calcium Signaling; Calcium ion; Calcium-Binding Proteins; Calmodulin; Cells; Complex; Computer Analysis; Cryoelectron Microscopy; Dissociation; EF Hand Motifs; Electrophysiology (science); Epilepsy; Feedback; Fluorescence; Gene Expression; Goals; Heart; Heterogeneity; Ion Channel; Learning; Link; Lobe; Mediating; Memory; Migraine; Modeling; Molecular Conformation; Molecular Structure; Mutation; Mutation Analysis; Neurons; Nuclear Magnetic Resonance; Physiological; Postsynaptic Membrane; Proteins; Regulation; Research; Retina; Role; Sampling; Signal Transduction; Site-Directed Mutagenesis; Structure; Techniques; Testing; Timothy syndrome; Work; X-Ray Crystallography; chronic pain; in vivo; insight; microcalorimetry; mutant; nervous system disorder; neuronal excitability; neurotransmission; postsynaptic neurons; prevent; response; sensor; transmission process; voltage

The overall objectives are to apply nuclear magnetic resonance (NMR) techniques in concert with experimental functional approaches to elucidate the molecular structure and regulatory mechanisms of neuronal L-type voltage-gated Ca2+ channel (CaV1.2) and its atomic-level structural and functional association with calcium binding protein-1 (CaBP1), -actinin1 (ACTN1) and calmodulin (CaM). During the next five years, we will use NMR, fluorescence, microcalorimetry, cryoEM, x-ray crystallography, and computational analysis to delineate the structure and dynamics of CaM and CaBP1 each bound to CaV1.2. Site-directed mutagenesis and electrophysiology functional analysis will be integrated with atomic– level structural information to understand how CaM, ACTN1 and CaBP1 each reciprocally control the Ca2+- dependent channel activity of CaV1.2 in neuronal functions. By pursuing these studies, we hope to gain an atomic-level understanding of how CaM, ACTN1 and CaBP1 each regulate CaV1.2 involved in controlling neuronal excitability and gene expression. In particular, we want to understand how protein target binding sites work in concert with calcium-binding sites to confer Ca2+-dependent channel activity of CaV1.2 at the postsynaptic membrane. This structural information will probe how L-type channel regulation may be connected to neurological disorders, including epilepsy, Alzheimer’s disease, and Timothy Syndrome. The Specific Aims are three-fold: (1) Determine the structural basis of CaV1.2 channel activation promoted by CaM and ACTN1; (2) Elucidate the structure and functional role of a CaM intermediate with 2 Ca2+ bound and determine its role in regulating Ca2+-dependent inactivation (CDI); (3) Determine the structural basis of how CaBP1 suppresses Ca2+-dependent inactivation (CDI) of CaV1.2.

总体目标是将核磁共振（NMR）技术应用于 与实验功能方法相结合，阐明分子结构和调控 神经元L型电压门控Ca2+通道（CaV1.2）的机制及其原子级结构和 与钙结合蛋白 1 (CaBP1)、α-肌动蛋白 1 (ACTN1) 和钙调蛋白 (CaM) 的功能相关。 在接下来的五年里，我们将使用核磁共振、荧光、微量热法、冷冻电镜、X射线晶体学、 和计算分析来描绘 CaM 和 CaBP1 各自结合的结构和动力学 CaV1.2。定点突变和电生理功能分析将与原子结合 水平结构信息，以了解 CaM、ACTN1 和 CaBP1 如何相互控制 Ca2+- CaV1.2 在神经元功能中的依赖性通道活性。通过进行这些研究，我们希望获得 对 CaM、ACTN1 和 CaBP1 如何各自调节参与控制的 CaV1.2 的原子水平理解 神经元兴奋性和基因表达。特别是，我们想了解蛋白质如何靶向 结合位点与钙结合位点协同作用，赋予 CaV1.2 的 Ca2+ 依赖性通道活性 在突触后膜处。该结构信息将探讨 L 型通道调节的方式 与神经系统疾病有关，包括癫痫、阿尔茨海默病和蒂莫西综合症。 具体目标有三个：（1）确定CaV1.2通道激活促进的结构基础 通过 CaM 和 ACTN1； (2) 阐明具有2个Ca2+的CaM中间体的结构和功能作用 结合并确定其在调节 Ca2+ 依赖性失活 (CDI) 中的作用； (3)确定结构 CaBP1 如何抑制 CaV1.2 的 Ca2+ 依赖性失活 (CDI) 的基础。