Molecular Determinants of L-type Calcium Channel Gating

描述(申请人提供)：钙离子通过质膜L型(CAV1.2)钙离子通道调节许多基本的生物学功能，包括肌肉收缩、激素释放和基因表达。L类通道功能表达失调是一系列疾病的基础，包括危及生命的房性心律失常、自闭症、夜盲和耳聋。此外，L类经络也是高血压、心绞痛、心律失常等严重危害公众健康的疾病的重要治疗靶点。然而，关于L类经络在各种疾病发病机制中的确切作用，人们的认识还存在很大差距，它们的全部治疗潜力在很大程度上仍未实现。僵局的一个关键因素是缺乏对L式通道、亚细胞靶向和门控行为背后的基本机制的洞察。我们的长期目标有两个：第一，增加对L型通道功能表达背后的结构-功能机制的基本分子理解；第二，将这些基本见解与对L型通道的(病理)生理作用和治疗潜力的新认识联系起来。我们结合重组和天然L型通道(Cav1.2)的电生理检测、蛋白质相互作用的FRET检测和通道亚基的免疫荧光检测有四个目的：1.阐明辅助Cav结构决定因素在重组L型通道A1C亚基的运输和门控调制中的作用。2.阐明与CAV？S相互作用的重要A1C结构决定因素，并确定它们在通道转运和门控调制中的作用。3.明确L类钙通道靶向心脏二元连接的结构决定因素。4.阐明蛋白激酶A调控心肌L型钙通道的分子决定因素及机制。

DESCRIPTION (provided by applicant): Ca2+ entry through plasma membrane L-type (Cav1.2) Ca2+ channels regulates many essential biological functions including muscle contraction, hormone release, and gene expression. Dysregulation of L-type channel functional expression underlies a wide range of diseases including life-threatening atrial arrhythmias, autism, night blindness, and deafness. Moreover, L-type channels are important therapeutic targets for a number of diseases that constitute a significant detriment to the public health such as hypertension, angina, and arrhythmias. Nevertheless, there are significant gaps in knowledge regarding the precise role of L-type channels in the pathogenesis of various diseases, and their full therapeutic potential remains largely unrealized. A key contributor to the impasse is lack of fundamental mechanistic insights into processes underlying L-type channel sub-cellular targeting and gating behavior. Our long-term objective is two fold: first, to increase fundamental molecular understanding of structure-function mechanisms underlying L-type channel functional expression; second, to bridge these basic insights to a new realization of the (patho)physiological roles and therapeutic potential of L-type channels. We combine electrophysiological assays of recombinant and native L-type (Cav1.2) channels, FRET detection of protein interactions, and immounofluorescence detection of channel subunits in 4 Aims: 1. Clarify the role of auxiliary Cav¿ structural determinants underlying trafficking and gating-modulation of recombinant L-type channel a1C subunits. 2. Clarify a1C structural determinants important for interacting with Cav¿s, and define their role in channel trafficking and gating-modulation. 3. Define the structural determinants underlying targeting of L-type Ca2+channels to dyadic junctions in heart. 4. Elucidate molecular determinants and mechanisms of protein kinase A modulation of cardiac L-type Ca2+channels.