Isoform-Specific Regulation of Skeletal Muscle Calcium Channel by Calmodulin

描述(申请人提供)：项目摘要/摘要本研究的目的是对钙调素(CaM)对骨骼肌钙释放通道(1型Ryanodine受体，RyR1)的调节机制有新的认识。RyR1是骨骼肌中主要的RyR亚型，负责在肌肉动作电位过程中从肌浆网释放钙，肌浆网是细胞内的钙储存库。RyR1是一种~550 kDa多肽的同源四聚体，受多种生理分子和蛋白质的调节，如ATP、钙离子、蛋白激酶和CaM等。已经证明结构域相互作用参与了RyR1的调节。虽然CaM结合位点在所有三种哺乳动物RyR亚型中都是保守的，但观察到了异构体依赖的调节。在亚微摩尔的钙离子浓度下，CaM激活RyR1，而抑制RyR2(心肌亚型)。保守的CaM结合区的相应点突变会不同地影响每种RyR亚型的CaM结合和CaM调节。这表明CaM的调控不是由CaM结合区参与的，而是由异构体特异性结构域参与的。RyR1结构域(类CaM结构域；CaMLD)被预测为类似于CaM的结构，与RyR1的CaM结合位点相互作用。这项研究中要检验的假设是，在亚微摩尔钙离子浓度下，CaM结合域和CaMLD之间的结构域间相互作用包括在RyR1特异的CaM激活中。CaMLD在RyRs异构体特异性CaM调控中的功能意义将通过两种途径来评估：(1)在CaMLD中表达重组RyR1/RyR2嵌合体通道和携带点突变的RyRs；(2)使用与RyR CaM结合域和CaMLD相对应的合成肽。我们将通过[~3H]ryanodine结合测量和单通道记录来对嵌合体和突变的RyRs进行功能分析。外源合成肽将被用来破坏结构域间的相互作用，这有望导致RyR活性的异构体依赖的变化。我们还将使用表面等离子体共振来测量CaM结合域和CaMLD之间的直接相互作用。此外，我们还将通过蛋白质覆盖和表面等离子共振筛选与CaMLD相互作用的RyR1额外结构域。这项研究将促进对参与RyR1钙调素调控的结构域复合体的理解。

DESCRIPTION (provided by applicant): Project Summary /Abstract The goal of the proposed research is to obtain new insights into the mechanism underlying regulation of the skeletal muscle Ca2+ release channel (type 1 ryanodine receptor, RyR1) by calmodulin (CaM), a cytoplasmic Ca2+ binding protein. RyR1 is the dominant RyR isoform in skeletal muscle and is responsible for the release of Ca2+ from the sarcoplasmic reticulum, an intracellular Ca2+ store, during muscle action potential. RyR1 is a homotetramer of ~550 kDa polypeptides and is regulated by various physiological molecules and proteins such as ATP, Ca2+, protein kinases and CaM. Involvement of domain interactions in regulation of RyR1 has been demonstrated. Although CaM binding site is conserved among all three mammalian RyR isoforms, isoform- dependent regulation is observed. RyR1 is activated, whereas RyR2 (cardiac muscle isoform) is inhibited by CaM at submicromolar Ca2+ concentrations. Corresponding point mutations in the conserved CaM binding domain differentially affect CaM binding and CaM regulation of each RyR isoform. This suggests that isoform- specific domains other than the CaM binding domain are involved in CaM regulation. A RyR1 domain (CaM- like domain; CaMLD), which was predicted to resemble the structure of CaM, was suggested to interact with the CaM binding site of RyR1. The hypothesis to be tested in the proposed research is that inter-domain interactions between the CaM binding domain and CaMLD is included in RyR1-specific CaM activation at submicromolar Ca2+ concentrations. Functional significance of CaMLD in isoform-specific CaM regulation of RyRs will be assessed by two approaches; (1) expression of recombinant RyR1/RyR2 chimera channels and mutant RyRs carrying point mutations in CaMLD, and (2) use of synthetic peptides corresponding with RyR CaM binding domain and CaMLD. We will perform functional analysis of chimera and mutant RyRs by [3H]ryanodine binding measurements and single channel recordings. Exogenous synthetic peptides will be used to disrupt inter-domain interactions, which is expected to result in isoform-dependent changes in RyR activity. We will also measure direct interactions between the CaM binding domain and CaMLD using surface plasmon resonance. In addition, we will screen an additional domain of RyR1 which interacts with CaMLD using protein overlays and surface plasmon resonance. The research will advance understanding of the domain complex involved in CaM regulation of RyR1.