DHPR DOMAINS CRITICAL TO EXCITATION-CONTRACTION COUPLING

摘要。该基金研究了二氢吡啶受体（DHPR）β 1a和β 2a亚基在骨骼肌和心肌兴奋-收缩（EC）偶联中的参与。响应于去极化，DHPR产生信号，该信号短暂地打开兰尼碱受体（RyR）通道，导致储存的Ca 2+的释放。由于DHPR和RyR表达的小鼠模型的可用性，在理解DHPR-RyR相互作用方面取得了相当大的进展。这些是缺乏α 2S的基因缺陷小鼠系和缺乏骨骼肌RyR 1同种型或缺乏骨骼肌DHPR β 1a亚基的基因敲除小鼠。拟议的实验将使用这些突变体来鉴定参与EC偶联的beta 1a和beta 2a亚基的结构域。β 1a的AC-末端区域，与结合α 1 S所需的BID结构域无关，将详细表征。这一β 1a的C-末端区域，与结合α 1 S所需的BID结构域无关，将详细表征。该C-末端可以引起DHPR和RyR的更强的共定位，或者对于产生打开RyR的信号是必需的。这两种可能性都将受到考验。为了解决这些问题，我们广泛使用双无效肌管，（α 1 S/β）-null和（β 1/RyR 1）-null，由小鼠育种产生。双空骨骼肌细胞应该允许在表达系统中研究α 2S/β和β/RyR相互作用，其中两个缺失的亚基可以表达和修饰。 申请的具体目标是：目标1。确定β 1a和β 2a在EC偶联特异性所需的DHPR表达中的作用;目的2。确定EC偶联所需的beta 1a的分子结构域;目的3。测试控制Ca 2+火花的功能性β-RyR相互作用;以及目标4。确定β是否触发Ca 2+瞬变的成分。后者通过表达缺乏II/III环的α 1 S构建体和通过在ES细胞衍生的心肌细胞中表达α 1C构建体来研究。主要方法包括a）在培养的单亚基缺陷或双缺失肌管中表达α 1、β和RyR亚基的cDNA构建体; B）β 1构建体的转基因过表达; c）在α 1C缺失心肌细胞中表达α 1C构建体; d）电压钳制细胞中Ca 2+电流和电荷运动的宏观测量;和e）Ca 2+瞬变和Ca 2+火花的共焦成像。DHPR-RyR相互作用对于理解骨骼肌和心肌正常和患病状态下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.