Structural and molecular requirements for DHPR and RyR1 bidirectional signaling

 描述(申请人提供)：骨骼肌收缩是由两个巨大的离子通道复合体，肌膜上的二氢吡啶受体(DHPR)和肌浆网(SR)中的兰尼定受体钙通道(RyR1)之间的物理相互作用启动的。DHPRα1S亚基是将该蛋白与RyR1偶联所必需的，但这种相互作用不可能在没有DHPRβ1a亚基的情况下发生，它在兴奋-收缩(EC)偶联中发挥着关键作用，但人们对此知之甚少。虽然这三种蛋白质对骨骼肌的功能都是必不可少的，但它们如何结合在一起在高等脊椎动物的三联体连接中产生EC偶联信号仍然是肌肉生物学最基本的悬而未决的问题之一。这项建议的长期目标是确定这些相互作用，并确定它们在正常和肌病条件下的相互关系。因此，我们在这里提出了一种创新的多学科方法，在其天然骨骼肌环境中对DHPR/RyR1复合体的结构/功能进行了系统的表征。在AIM-1中，我们提出了一个新的DHPR络合物之间的缔合模型。在这里，我们将测试β1a和α1S亚基的亮氨酸拉链基序在连接相邻的DHPR颗粒和EC偶联信号中的作用。这些研究将使用多学科方法，包括定点突变、钙成像、全细胞膜片钳和小鼠培养的肌管的冷冻断裂分析。在AIM-2中，我们将使用一种创新的基于FRET的方法来定位完整肌管中DHPR复合体的关键结构域相对于彼此的位置(S)。我们还将确定DHPR复合体的结构如何受到关键亮氨酸拉链中断的影响，以及它如何 在正常和病理生理条件下(恶性高热综合征)的EC偶联过程中进行调整。在AIM-3中，我们将使用我们基于FRET的分析来确定DHPR复合体与RyR1在EC偶联中所涉及的关键功能域之间的关系。这些研究将确定两个通道之间的物理相互作用位置，并将有助于确定DHPR和RyR1的相对方向，直接测试我们的工作模型。这一建议的成功完成将提供骨架型EC偶联所需的关键分子间相互作用的详细结构图，从而为理解这些通道在健康和疾病中的物理偶联提供必要的信息。

 DESCRIPTION (provided by applicant): Skeletal muscle contraction is initiated by a proposed physical interaction between two enormous ion channel complexes, the dihydropyridine receptor (DHPR) in the sarcolemma and the ryanodine receptor Ca2+ channel (RyR1) in the sarcoplasmic reticulum (SR). The DHPR α1S subunit is essential to couple this protein to RyR1, but this interaction cannot occur without the DHPR β1a subunit, which plays a pivotal but poorly understood role in excitation- contraction (EC) coupling. While all three of these proteins are absolutely required for skeletal muscle function, how they fit together to produce the EC-coupling signal in the triad junction of higher vertebrate remains as one of the most fundamental unanswered question of muscle biology. The long-term goal of this proposal is to identify these interactions and define their interrelationship under normal and myopathic conditions. Consequently, here we propose a systematic structure/function characterization of the DHPR/RyR1 complex in its native skeletal muscle environment using an innovative multidisciplinary approach. In Aim-1 we propose a new model of association between DHPR complexes. Here we will test the role of leucine zipper motifs of β1a and α1S subunit in both interlinking adjacent DHPR particles and in EC- coupling signaling. These studies will use a multi-disciplinary approach involving site-directed mutagenesis, Ca2+ imaging, whole-cell patch clamp and freeze-fracture analyses in mouse cultured myotubes. In Aim-2 we will use an innovative FRET-based approach to map the position(s) of critical domains of the DHPR complex relative to each other within intact myotubes. We will also determine how the structure of the DHPR complex is affected by the disruption of the critical leucine zippers as well as how it adjusts during EC-coupling under normal and pathophysiological conditions (malignant hyperthermia syndrome). In Aim-3 we will use our FRET-based assay to determine the orientation of the DHPR complex in relationship to key functional domains of RyR1 implicated in EC coupling. These studies will both identify sites of physical interaction between the two channels and will help to determine the relative orientation of the DHPR and RyR1, directly testing our working model. Successful completion of this proposal should provide with a detailed structural map of critical inter-molecular interactions required for skeletal-type EC-coupling, therefore, provide with essential information to understand physical coupling between these channels in health and disease.