Structural and molecular requirements for DHPR and RyR1 bidirectional signaling

 描述（由申请人提供）：骨骼肌收缩由两种巨大离子通道复合物（肌膜中的二氢吡啶受体（DHPR）和肌浆网（SR）中的兰尼碱受体Ca 2+通道（RyR 1））之间的拟议物理相互作用引发。DHPR α 1 S亚基是将该蛋白偶联至RyR 1所必需的，但如果没有DHPR β1a亚基，这种相互作用就无法发生，DHPR β1a亚基在兴奋-收缩（EC）偶联中起着关键但知之甚少的作用。虽然所有这三种蛋白质都是骨骼肌功能所必需的，但它们如何组合在一起以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中最基本的未回答的问题之一。本提案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，在这里，我们提出了一个系统的结构/功能表征的DHPR/RyR 1复合物在其天然骨骼肌环境中使用创新的多学科方法。在Aim-1中，我们提出了DHPR复合物之间缔合的新模型。在这里，我们将测试β1a和α 1 S亚基的亮氨酸拉链基序在连接相邻DHPR颗粒和EC偶联信号传导中的作用。这些研究将使用多学科的方法，包括定点诱变，Ca 2+成像，全细胞膜片钳和冷冻断裂分析在小鼠培养的肌管。在Aim-2中，我们将使用一种创新的基于FRET的方法来绘制完整肌管内DHPR复合物的关键结构域相对于彼此的位置。我们还将确定DHPR复合物的结构是如何受到关键亮氨酸拉链破坏的影响，以及它如何影响DHPR复合物的结构。 在正常和病理生理条件下（恶性高热综合征）的EC偶联过程中进行调节。在目的-3，我们将使用我们的FRET为基础的测定，以确定方向的DHPR复合物的关系，涉及EC耦合的RyR 1的关键功能域。这些研究将确定两个通道之间的物理相互作用位点，并有助于确定DHPR和RyR 1的相对方向，直接测试我们的工作模型。成功完成本提案应提供一个详细的结构图的关键分子间相互作用所需的神经元型EC耦合，因此，提供必要的信息，以了解这些通道之间的物理耦合在健康和疾病。

 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.