DHPR DOMAINS CRITICAL TO EXCITATION-CONTRACTION COUPLING

抽象的。这项资助研究了二氢吡啶受体(DHPR)β1a和β2a亚单位在骨骼肌和心肌兴奋-收缩(EC)偶联中的参与。作为对去极化的反应，DHPR产生一个信号，短暂地打开Ryanodine受体(RyR)通道，导致储存的钙离子释放。随着表达DHPR和RyR的小鼠模型的出现，在理解DHPR-RyR相互作用方面已经取得了相当大的进展。这些是缺乏Alpha2S的不育小鼠和缺乏骨骼肌RyR1亚型或缺乏骨骼肌DHPR的Beta1a亚单位的基因敲除小鼠。拟议的实验将使用这些突变体来识别参与EC偶联的Beta1a和Beta2a亚单位的结构域。将详细描述Beta1a的AC-末端区域，该区域与与α1S结合所需的Bid结构域无关。将详细描述Beta1a的这个C-末端区域，它与与α1S结合所需的BID结构域无关。这个C末端可以带来DHPR和RyR的更强的共定位，或者对于打开RyR的信号的产生是必不可少的。这两种可能性都将得到测试。为了解决这些问题，我们广泛使用了通过小鼠繁殖产生的双零肌管，(α1S/β)-零和(β1/RyR1)-零。双零骨骼肌细胞应该可以在表达系统中研究α2S/β和β/RyR的相互作用，在这个表达系统中，两个缺失的亚基可以被表达和修饰。应用的具体目的是：目的1.确定Beta1a和Beta2a在EC偶联所需的DHPR表达中的作用；目的2.确定EC偶联所需的Beta1a的分子结构域；目的3.测试控制钙火花的功能β-RyR相互作用；以及目的4.确定β是否触发钙瞬变的一个组成部分。后者是通过缺乏II/III环的Alpha1S结构的表达和ES细胞来源的心肌细胞Alpha1C结构的表达来研究的。主要方法包括：a)在培养的单亚基缺失或双缺失的肌管中表达α1、β和RyR亚单位的c DNA结构；b)转基因过表达β1结构；c)在α1C缺失的心肌细胞中表达α1C结构；d)对电压钳制细胞中的钙电流和电荷运动进行宏观测量；以及e)钙瞬变和钙火花的共聚焦成像。DHPR-RyR相互作用对于了解骨骼肌和心肌正常和疾病状态下EC偶联的分子基础至关重要。

ABSTRACT. This grant examines the participation of the dihydropyridine receptor (DHPR) beta1a and beta2a subunits in skeletal and cardiac muscle excitation-contraction (EC) coupling. In response to depolarization, the DHPR products a signal that briefly opens ryanodine receptor (RyR) channels leading toe the release of stored Ca2+. Considerable progress in the understanding DHPR-RyR interactions has been made by the availability of mouse models for the expression of DHPRs and RyRs. These are the dysgenic mouse line lacking alpha2S and knockout mice lacking the skeletal muscle RyR1 isoform or lacking the beta1a subunit of the skeletal muscle DHPR. The proposed experiments will use these mutants to identify domains of the beta1a and beta2a subunits that participate in EC coupling. AC-terminus region of beta1a, unrelated to the BID domain required for binding to alpha1S, will be characterized in detail. This C-terminus region of beta1a, unrelated to the BID domain required for binding to alpha1S, will be characterized in detail. This C- terminus could bring about a stronger colocalization of DHPRs and RyRs or could be essential for the generation of the signal that opens the RyR. Both possibilities will be tested. To address these questions, we make extensive use of double-null myotubes, (alpha1S/beta)-null and (beta1/RyR1)-null, generated by mouse breeding. Double-null skeletal muscle cells should permit studies of alpha2S/beta and beta/RyR interactions in expression systems in which the two missing subunits can be expressed and modified. The specific aims of the application are: Aim 1. Establish the role of beta1a and beta2a in the expression of DHPRs specifically required for EC coupling; Aim 2. Identify molecular domains of beta1a required for EC coupling; Aim 3. Test functional beta-RyR interactions controlling Ca2+ sparks; and Aim 4. Determine whether beta triggers a component of the Ca2+ transient. The latter is investigated by expression of alpha1S constructs lacking the II/III loop and by expression of alpha1C constructs in ES cell-derived cardiomyocytes. The main methods include a) expression of cDNA constructs of alpha1, beta and RyR subunits in single subunit-deficient or in double-null myotubes in culture; b) transgenic over-expression of beta1 constructs; c) expression of alpha1C constructs in alpha1C-null cardiomyocytes; d) macroscopic measurements of Ca2+ currents and charge movements in voltage- clamped cells; and e) confocal imaging of Ca2+ transients and Ca2+ sparks. DHPR-RyR interactions are crucial for understanding the molecular basis of EC coupling in normal and diseased states of skeletal and cardiac muscle.