Regulation of Membrane Excitability

项目摘要 在许多细胞类型中，质膜中的电压门控Ca 2+通道通过以下途径触发细胞内Ca 2+释放： 内质网中的兰尼碱受体（RyRs）。在骨骼肌中，一种特殊的 发生信号传导，使得CaV1.1（肌肉Ca通道的主要亚基，也称为 2个以上 DHPR）通过构象相互作用激活RyR 1（骨架RyR亚型）。我们的长期目标 研究的目的是确定这种构象相互作用所需的最小蛋白质组，并确定 它们相互作用的场所。根据以前的工作和我们自己的初步结果，我们知道， 除了CaV1.1和RyR 1之外，还需要另外两种蛋白质：“Stac”衔接蛋白和junctophilin。 所有四种蛋白质（CaV，RyR，Stacs和junctophilins）的不同亚型也在神经细胞中表达。 系统和CaV、RyR和junctophilins在心脏中表达。值得注意的是，CaV、RyR和 嗜连接蛋白引起骨骼肌、心脏和神经系统的遗传性人类疾病。明确 了解这些疾病的发病机制将受益于更好地了解这些疾病是如何发生的。 蛋白质之间相互作用，而所提出的实验的一个主要焦点就是实现这一点 认识为此，我们已经开发了一些实验工具，包括一个构建 仅编码RyR 1的胞质结构域（含有RyR 1残基1-4300但缺乏 约700个C-末端残基，形成肌浆网中的离子传导孔和锚RyR 1）。 此外，我们已经建立了在非肌肉细胞中获得高水平表达CaV1.1的能力。与 这些和其他结果证明了可行性，我们建议使用肌肉和非肌肉中的cDNA表达， 肌肉细胞，然后通过膜片钳，共聚焦荧光显微镜，生物化学和电子显微镜 显微镜追求以下具体目标。 目标1.使用tsA 201细胞中的表达来定义骨骼、心脏和骨骼肌之间相互作用的决定因素。 以及Stac蛋白、亲连接蛋白和RyR的神经元同种型。 目标二。为了利用tsA 201和肌细胞的不同方法优势来探索 CaV1.1和RyR 1之间信号传导的分子决定因素。肌管将用于检测Stac亚型 支持EC偶联的特异性和测试DHPR β 1亚基的作用。TSA 201细胞将用于 确定突变如何影响CaV1.1门控电荷运动，以及肌管探测 EC耦合装置的组成部分。 目标3：为了确定CaV1.1-RyR 1功能相互作用是否可以在tsA 201细胞中重建， 部分具有RyR 1cyto或完全具有全长RyR 1。

Project Summary In many cell types, voltage-gate Ca2+ channels in the plasma membrane trigger intracellular Ca2+ release via ryanodine receptors (RyRs) in the endoplasmic reticulum. In skeletal muscle, a specialized form of this signaling occurs such that CaV1.1 (the principle subunit of the muscle Ca channel, also referred to as the 2+ DHPR) activates RyR1 (the skeletal RyR isoform) via a conformational interaction. A long-term goal of our research is to identify the minimum set of proteins needed for this conformational interaction, and to determine their sites of interaction with one another. Based on previous work and our own preliminary results we know that in addition to CaV1.1 and RyR1, two other proteins are required: a “Stac” adaptor protein and junctophilin. Varying isoforms of all four proteins (CaVs, RyRs, Stacs and junctophilins) are also expressed in the nervous system and CaVs, RyRs and junctophilins are expressed in heart. Significantly, mutations of CaVs, RyRs and junctophilins give rise to inherited, human disorders of skeletal muscle, heart and the nervous system. Clearly understanding the pathogenesis of such disorders would benefit from a better understanding of how these proteins interact with one another, and a major focus of the proposed experiments is to achieve this understanding. Toward this end, we have developed a number of experimental tools, including a construct encoding only the cytoplasmic domain of RyR1 (“RyR1cyto” containing RyR1 residues 1-4300 but lacking the ~700 C-terminal residues that form the ion conducting pore and anchor RyR1 in the sarcoplasmic reticulum). Additionally, we have established the ability to obtain high level expression of CaV1.1 in non-muscle cells. With these and other results demonstrating feasibility, we propose to use cDNA expression in muscle and non- muscle cells, followed by patch clamping, confocal fluorescence microscopy, biochemistry and electron microscopy to pursue the following specific aims. Aim 1. To use expression in tsA201 cells to define the determinants for interactions between skeletal, cardiac and neuronal isoforms of the Stac proteins, the junctophilins, and RyRs. Aim 2. To exploit the differential methodological strengths of tsA201 and muscle cells for probing the molecular determinants of signaling between CaV1.1 and RyR1. Myotubes will be used to test the Stac isoform specificity for support of EC coupling and to test the role of the DHPR 1 subunit. tsA201 cells will be used to determine how mutations affect CaV1.1 gating charge movements, and myotubes to probe proximities among constituents of the EC coupling apparatus. Aim 3. To determine whether CaV1.1-RyR1 functional interactions can be reconstituted in tsA201 cells, either partially with RyR1cyto or entirely with full-length RyR1.