Tuning of CaV channel dynamics by stac proteins

stac 蛋白调节 CaV 通道动力学

基本信息

批准号：
10471966
负责人：
Manu Ben Johny
金额：
$ 35.1万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-15 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10471966
关键词：
Action Potentials Acute Adaptor Signaling Protein Affinity Age Alternative Splicing Binding Biological Biological Assay Brain Calcineurin Cells Childhood Schizophrenia Complex Cysteine-Rich Domain Data Dependence Development Down-Regulation Electrophysiology (science)Event Flow Cytometry Fluorescence Resonance Energy Transfer Formulation Functional disorder Future Genetic Harvest Light Link Mediating Membrane Modeling Molecular Morphology Muscle Mutagenesis Mutation Neurobiology Neurons Noise Optics Parkinson Disease Pathogenesis Phosphorylation Physiological Physiology Post-Translational Protein Processing Probability Process Property Protein Isoforms Proteins RNA RNA Editing RNA Interference Regulation Role Scheme Signal Transduction Skeletal Muscle Substantia nigra structure Surface System Testing Therapeutic Up-Regulation Variant Work calmodulin-dependent protein kinase II congenital myopathy cytotoxicity dopaminergic neuron frontier genetic regulatory protein in silico innovation insight overexpression pars compacta prevent small molecule stoichiometry therapeutic target tool trafficking voltage clamp

项目摘要

Selective degeneration of dopaminergic (DA) neurons of substantia nigra pars compacta (SNc) is a critical event in the progression of Parkinson’s disease. These neurons are vulnerable as they undergo autonomous pacemaking with an unusual reliance on Ca2+-influx via CaV1.3 channels. Unlike other pacemaking neurons that remain relatively protected during PD pathogenesis, SNc neurons have larger Ca2+-currents with blunted inactivation, thus aggravating the propensity for Ca2+-overload. Given its central role, identifying regulatory proteins that tune Ca2+-channel function in SNc neurons is critical. One attractive regulatory candidate is SH3 and cysteine rich domain (stac) protein, a muscle and neuron specific protein that has emerged as a requisite component of skeletal muscle excitation-contraction machinery and as a locus for congenital myopathies. Even so, the neuronal functions of stac remains poorly defined, although stac2- mutations have been linked to childhood-onset schizophrenia. Motivated by preliminary data showing both the presence of stac in SNc neurons, and its functional role to selectively diminish Ca2+/CaM-dependent inactivation (CDI) in heterologous systems, we here dissect the molecular functions of stac, underlying mechanisms, and potential role in tuning SNc pacemaking, utilizing a bevy of molecular tools and quantitative optical and electrophysiological approaches. This project will usher in an exciting era of discovery via three specific aims. (1) How do stac isoforms tune CaV function? We seek to develop a unified mechanistic scheme for stac regulation of CaV1.3, by incorporating effects of alternative-splicing and RNA-editing, and by rigorous quantification of stac effects on CDI, baseline channel activity (PO), and surface-membrane trafficking (Nmem). (2) What are molecular determinants and mechanisms that support and refine stac regulation of CaV1? Informed by our prior mechanistic study, we seek to elucidate the molecular underpinnings for stac-regulation of CaV1.3 PO and Nmem and whether physiological or pathophysiological processes may fine-tune stac modulation. (3) How does multiprong- modulation of CaV1 by stac tune SNc pacemaking and Ca2+ signaling? Here, we elucidate whether either downregulation or upregulation of stac2 tunes endogenous CaV1 leading to alterations in pacemaking and action potential morphology of SNc neurons. This project promises unprecedented progress in elucidating multifaceted mechanisms for stac regulation of CaV1, an emerging frontier for Ca2+-channel physiology. Furthermore, this work may shed new insights into regulatory process that sculpt Ca2+-influx via CaV1.3 into SNc neurons, an important vulnerability for PD pathogenesis and a potentially promising therapeutic target.

黑质致密部 (SNc) 多巴胺能 (DA) 神经元的选择性变性是帕金森病进展中的一个关键事件，因为这些神经元很脆弱。经历自主起搏，异常依赖通过 CaV1.3 通道的 Ca2+ 流入。与在 PD 发病过程中保持相对保护的其他起搏神经元不同，SNc 神经元具有较大的 Ca2+ 电流且钝化失活，从而加剧了鉴于 Ca2+ 超载的核心作用，识别调节 Ca2+ 通道的调节蛋白。 SNc 神经元的功能至关重要，SH3 和富含半胱氨酸是一种有吸引力的调节候选者。结构域（stac）蛋白，一种肌肉和神经元特异性蛋白，已成为必需的蛋白骨骼肌兴奋-收缩机制的组成部分，并作为先天性运动的场所即便如此，尽管 stac2-，stac 的神经元功能仍然不清楚。初步数据表明突变与儿童期发病的精神分裂症有关。显示 SNc 神经元中 stac 的存在及其选择性减弱的功能作用异源系统中 Ca2+/CaM 依赖性失活 (CDI)，我们在此剖析分子 stac 的功能、基本机制以及在调整 SNc 起搏中的潜在作用，利用一系列分子工具以及定量光学和电生理学方法。 (1) stac亚型如何调整 CaV 功能？我们寻求开发一个统一的 stac 调节机械方案 CaV1.3，通过结合选择性剪接和 RNA 编辑的作用，并通过严格的量化 stac 对 CDI、基线通道活动 (PO) 和表面膜的影响 (Nmem) (2) 支持和完善的分子决定因素和机制是什么。根据我们之前的机制研究，我们试图阐明 CaV1 的 stac 调控？ CaV1.3 PO 和 Nmem 的 stac 调节的分子基础，以及生理或 (3) 多管齐下如何通过 stacune SNc 起搏和 Ca2+ 信号调节 CaV1 在这里，我们阐明是否 stac2 的下调或上调会调节内源性 CaV1，从而导致该项目有望研究 SNc 神经元的起搏和动作电位形态。在阐明 Stac 调控 CaV1 的多方面机制方面取得了前所未有的进展，CaV1 是一种此外，这项工作可能会为 Ca2+ 通道生理学的新兴前沿提供新的见解。通过 CaV1.3 将 Ca2+ 流入 SNc 神经元的调节过程，这是一个重要的脆弱性 PD 发病机制和潜在有前途的治疗靶点。