DHPR beta subunit and excitation-contraction coupling

描述（由申请人提供）：该资助研究了DHPR β 1a亚基在骨骼肌兴奋-收缩耦合中的参与。核心假设是DHPR β 1a亚基与RyR1之间的蛋白-蛋白相互作用是EC耦合过程中DHPR向RyR1通道传递信号所必需的。该假设通过体外重组蛋白和基于细胞的方法的结合来验证。使用FRET接近确定用于研究提出的beta1a结构模型。重组蛋白和体外表达方法用于绘制β 1a和RyR1中相互结合的结构域和分子基序。电生理方法在原代培养的beta1 KO和RyR1 KO小鼠肌管中实施，研究了beta1a和RyR1相互作用的功能后果。Aim 1将基于beta1a与MAGUK蛋白PSD-95的同源性建立beta1a结构域组织模型。我们建议在融合CFP作为供体和共价结合FIAsH作为受体的beta1a变体中使用FRET来研究beta1a的结构域组织。目的2将研究β 1a的c端结构域在EC耦合中的作用。基于细胞的诱变和体外下拉策略用于评估β 1a中存在的C端和n端七肽重复序列的功能意义。Aim 3将描述RyR1结合beta1a的一个关键区域，并确定beta1a-RyR1相互作用的功能相关性。基于细胞的诱变和体外下拉策略将确定位于RyR1 3490-3523区域的beta1a推定结合位点的重要性。beta1a/RyR1可能导致DHPR和RyR1的强对接，并且/或者可能对打开RyR1的信号的产生至关重要。这两种功能虽然看似不同，但可能代表了同一分子相互作用的两种表现。本应用程序的目的是获得DHPR β 1a如何与RyR1相互作用的详细分子洞察力，并追求这种相互作用的功能后果。这一信息对于理解正常EC偶联的分子基础以及EC偶联剧烈改变的病变状态的分子基础至关重要。

DESCRIPTION (provided by applicant): This grant examines the participation of the DHPR beta1a subunit in excitation-contraction coupling in skeletal muscle. The central hypothesis is that protein-protein interactions between the DHPR beta1a subunit and RyR1 are essential for signals transmitted from the DHPR to the RyR1 channel during EC coupling. The hypothesis is tested by a combination of in-vitro recombinant protein and cell-based approaches. Proximity determinations using FRET are used to investigate a structural model proposed for beta1a. Recombinant protein and in-vitro expression approaches are used to map domains and molecular motifs in beta1a and RyR1 that bind to each other. Electrophysiological approaches implemented in primary cultured myotubes from beta1 KO and RyR1 KO mice investigate the functional consequences of the interaction of beta1a and RyR1. Aim 1 will develop a model of the domain organization of beta1a based on its homology to the MAGUK protein PSD-95. We propose to investigate the domain organization of beta1a using FRET in beta1a variants with fused CFP as the donor and covalently-bound FIAsH as the acceptor. Aim 2 will examine the role of the C-terminal domain of beta1a in EC coupling. Cell-based mutagenesis and in-vitro pull-down strategies are used to assess the functional significance of C- and N-terminal heptad repeats present in beta1a. Aim 3 will characterize a critical region of RyR1 that binds beta1a and determine functional correlates of the beta1a-RyR1 interaction. Cell-based mutagenesis and in-vitro pull-down strategies will determine the significance of a putative binding site for beta1a located in the 3490-3523 region of RyR1. A beta1a/RyR1 could bring about strong docking of the DHPR and RyR1, and/or could be essential to the generation of the signal that opens RyR1. These two functions, although seemingly dissimilar, may represent two manifestations of the same molecular interaction. The purpose of this application is to gain a detailed molecular insight on how DHPR beta1a interacts with RyR1, and to pursue the functional consequences of this interaction. This information is crucial for understanding the molecular basis of normal EC coupling as well as the molecular basis of diseased states in which EC coupling is drastically altered.