Isoform-Specific Regulation of Skeletal Muscle Calcium Channel by Calmodulin

描述（由申请人提供）：项目摘要/摘要拟议研究的目标是获得钙调蛋白（CaM）（一种细胞质Ca 2+结合蛋白）对骨骼肌Ca 2+释放通道（1型兰尼碱受体，RyR 1）调节机制的新见解。RyR 1是骨骼肌中占优势的RyR亚型，负责在肌肉动作电位期间从肌浆网（细胞内Ca 2+库）释放Ca 2+。RyR 1是约550 kDa多肽的同源四聚体，受各种生理分子和蛋白质如ATP、Ca 2+、蛋白激酶和CaM的调节。已经证明了结构域相互作用参与RyR 1的调节。虽然CaM结合位点在所有三种哺乳动物RyR同工型中是保守的，但观察到同工型依赖性调节。RyR 1被激活，而RyR 2（心肌亚型）被CaM在亚微摩尔Ca 2+浓度下抑制。在保守的钙调素结合结构域的相应点突变差异影响钙调素结合和钙调素调节每个RyR亚型。这表明，异构体特异性结构域以外的钙调素结合结构域参与钙调素的调节。RyR 1结构域（CaM-like domain; CaMLD）被预测为类似于CaM的结构，被认为与RyR 1的CaM结合位点相互作用。在拟议的研究中要测试的假设是，钙调素结合结构域和CaMLD之间的域间相互作用包括在RyR 1特异性钙调素激活在亚微摩尔Ca 2+浓度。将通过两种方法评估CaMLD在RyR的同种型特异性CaM调节中的功能意义;（1）表达重组RyR 1/RyR 2嵌合体通道和携带CaMLD中的点突变的突变体RyR，以及（2）使用与RyR CaM结合结构域和CaMLD对应的合成肽。我们将通过[3 H]ryanodine结合测量和单通道记录对嵌合体和突变体RyRs进行功能分析。外源性合成肽将用于破坏结构域间的相互作用，预计这将导致RyR活性的亚型依赖性变化。我们还将使用表面等离子体共振测量CaM结合结构域和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RyR 1的另外的结构域。这项研究将促进对RyR 1的CaM调控所涉及的结构域复合物的理解。

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.