Structural and molecular requirements for DHPR and RyR1 bidirectional signaling

DHPR 和 RyR1 双向信号传导的结构和分子要求

• 批准号: 9029525
• 负责人: Claudio F Perez
• 金额: $ 47.65万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-16 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9029525
• 关键词: Affect; Beryllium; Binding; Biological; Biological Assay; Biology; C-terminal; Cells; Complex; Coupling; Development; Dihydropyridine Receptors; Disease; Electrophysiology (science); Environment; Fluorescence Resonance Energy Transfer; Freeze Fracturing; Future; Goals; Health; Image; In Situ; Individual; Ion Channel; Leucine Zippers; Link; Malignant hyperpyrexia due to anesthesia; Maps; Measures; Methods; Modeling; Molecular; Monitor; Mus; Muscle; Muscle Contraction; Muscle Fibers; Muscle function; Mutagenesis; Mutation; Myopathy; Pharmaceutical Preparations; Play; Positioning Attribute; Proteins; Public Health; Role; RyR1; Ryanodine Receptor Calcium Release Channel; Sarcolemma; Sarcoplasmic Reticulum; Signal Transduction; Site; Site-Directed Mutagenesis; Skeletal Muscle; Structure; Syndrome; Tail; Techniques; Testing; Triad Acrylic Resin; Work; base; crosslink; disease-causing mutation; human disease; in vivo; innovation; interdisciplinary approach; novel; novel therapeutics; particle; patch clamp; public health relevance; research study; skeletal; spatial relationship; therapeutic target

 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.