L-type Ca2+ Channel Regulation by Calmodulin and CaBP1

总体目标是将核磁共振（NMR）技术应用于 与实验功能方法相一致，以阐明分子结构和调节 神经元L型电压门控钙通道（CaV1.2）的机制及其原子水平的结构和功能 与钙结合蛋白1（CaBP 1）、β-辅肌动蛋白1（ACTN 1）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，冷冻电镜，X射线晶体学， 和计算分析来描绘CaM和CaBP 1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- 水平的结构信息，以了解CaM，ACTN1和CaBP1各自如何控制Ca2 +- CaV1.2在神经元功能中的依赖性通道活性。通过开展这些研究，我们希望获得 在原子水平上理解CaM、ACTN1和CaBP1如何各自调节参与控制的CaV1.2 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的钙依赖性通道活性 在突触后膜上这一结构信息将探讨L型通道调节可能是如何 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征。 具体目的有三：（1）确定CaV1.2通道激活促进的结构基础 （2）阐明了一个含2个Ca~（2+）的CaM中间体的结构和功能 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 CaBP 1如何抑制CaV1.2的Ca2+依赖性失活（CDI）的基础。

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.