DHPR DOMAINS CRITICAL TO EXCITATION-CONTRACTION COUPLING

摘要。该资助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。为了响应去极化，DHPR产生一个信号，该信号短暂地打开了ryanodine受体（RyR）通道，导致储存的Ca2+释放。在了解DHPR-RyR相互作用方面取得了相当大的进展，这是由于dhpr和ryr表达的小鼠模型的可用性。这些是缺乏alpha2S的基因异常小鼠系和缺乏骨骼肌RyR1亚型或缺乏骨骼肌DHPR的β 1a亚基的基因敲除小鼠系。提出的实验将使用这些突变体来识别参与EC耦合的beta1a和beta2a亚基的结构域。beta1a的交流端区域与与alpha1S结合所需的BID结构域无关，将被详细表征。beta1a的c端区域与与alpha1S结合所需的BID结构域无关，将被详细描述。这个C末端可能导致dhpr和RyR更强的共定位，或者可能对打开RyR的信号的产生至关重要。这两种可能性都将受到考验。为了解决这些问题，我们广泛使用了小鼠育种产生的双空肌管(alpha1S/beta)-null和(beta1/RyR1)-null。双空骨骼肌细胞应该允许在两个缺失亚基可以表达和修饰的表达系统中研究α 2s / β和β /RyR相互作用。该应用程序的具体目标是：目标1。建立beta1a和beta2a在EC偶联特异性dhpr表达中的作用；目标2。确定EC偶联所需的beta1a分子结构域；目标3。测试功能β - ryr相互作用控制Ca2+火花；和Aim 4。确定β是否触发Ca2+瞬态的一个组成部分。后者是通过缺乏II/III环的alpha1S构建体的表达和ES细胞源性心肌细胞中alpha1C构建体的表达来研究的。主要方法有：a)在培养中单亚基缺失或双亚基缺失的肌管中表达α 1、β和RyR亚基cDNA构建体；B)转基因过表达beta1构建体；c) alpha1C构建体在无alpha1C心肌细胞中的表达；d)电压箝位细胞中Ca2+电流和电荷运动的宏观测量；e) Ca2+瞬态和Ca2+火花的共聚焦成像。DHPR-RyR相互作用对于理解骨骼肌和心肌正常和病变状态下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.