Isoform-Specific Regulation of Skeletal Muscle Calcium Channel by Calmodulin

项目概述/摘要本研究的目的是通过钙调蛋白（calmodulin, CaM）（一种胞质Ca2+结合蛋白）对骨骼肌Ca2+释放通道（1型ryanodine受体，RyR1）的调控机制获得新的见解。RyR1是骨骼肌中主要的RyR亚型，负责在肌肉动作电位期间从肌浆网释放Ca2+，这是细胞内Ca2+储存。RyR1是~550 kDa多肽的同聚体，受ATP、Ca2+、蛋白激酶和CaM等多种生理分子和蛋白的调控。结构域相互作用参与RyR1的调控已被证实。尽管CaM结合位点在所有三种哺乳动物的RyR异构体中都是保守的，但观察到异构体依赖性调节。RyR1被激活，而RyR2（心肌异构体）在亚微摩尔Ca2+浓度下被CaM抑制。CaM结合域的相应点突变对各RyR异构体的CaM结合和CaM调控有不同的影响。这表明除了CaM结合结构域外，还存在异构体特异性结构域参与CaM调控。RyR1结构域（CaM- like domain, CaMLD）与RyR1的CaM结合位点相互作用，该结构域与CaM的结构相似。在本研究中需要验证的假设是，CaM结合域和CaMLD之间的域间相互作用包括在亚微摩尔Ca2+浓度下ryr1特异性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方法进行评估；(1)在CaMLD中表达重组RyR1/RyR2嵌合体通道和携带点突变的突变体RyRs，(2)合成与RyR CaM结合域和CaMLD对应的肽。我们将通过[3H]ryanodine结合测量和单通道记录对嵌合体和突变型RyRs进行功能分析。外源性合成肽将被用来破坏结构域间的相互作用，这将导致RyR活性的异构体依赖性变化。我们还将使用表面等离子体共振测量CaM结合域和CaMLD之间的直接相互作用。此外，我们将使用蛋白质覆盖和表面等离子体共振来筛选与CaMLD相互作用的RyR1的附加结构域。该研究将促进对涉及RyR1的CaM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.