Goonasekera Sanjeewa A, Beard Nicole A, Groom Linda, Kimura Takashi, Lyfenko Alla D, Rosenfeld Andrew, Marty Isabelle, Dulhunty Angela F, Dirksen Robert T
Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA.
J Gen Physiol. 2007 Oct;130(4):365-78. doi: 10.1085/jgp.200709790. Epub 2007 Sep 10.
Ca(2+) release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca(2+) to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation-contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca(2+) release during excitation-contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca(2+) release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca(2+) release during excitation-contraction coupling in skeletal muscle.
细胞内钙库的钙离子（Ca(2+)）释放受多种蛋白质间复杂相互作用的调控。三联蛋白是横纹肌连接肌质网的一种跨膜糖蛋白，它与肌钙蛋白和1型兰尼碱受体（RyR1）相互作用，将管腔Ca(2+)的变化传递给释放机制。然而，三联蛋白与RyR1的结合在骨骼肌兴奋 - 收缩偶联中的潜在影响仍不清楚。在此我们表明，三联蛋白与RyR1的结合对于兴奋 - 收缩偶联期间的快速Ca(2+)释放至关重要。为了评估三联蛋白 - RyR1相互作用的功能影响，我们在RyR1基因缺失（患肌营养不良）的肌管中表达了RyR1突变体，其中RyR1腔内末端环中的三个带负电荷残基（D4878、D4907和E4908）中的一个或多个被突变为丙氨酸。免疫共沉淀显示，在三联（D4878A/D4907A/E4908A）突变体和其中一个双突变体（D4907A/E4908A）中，三联蛋白而非连接蛋白与RyR1的结合被消除，在D4878A/D4907A双突变体中部分降低，但不受单个（D4878A、D4907A、E4908A）突变或D4878A/E4908A双突变的影响。功能研究表明，电压门控和配体门控的肌质网Ca(2+)释放速率与三联蛋白结合中断的程度成比例降低。在不存在三联蛋白的情况下，对野生型和三联突变体通道进行的兰尼碱结合、单通道记录和钙释放实验表明，腔内环突变不会直接改变RyR1的功能。这些发现表明，连接蛋白和三联蛋白与RyR1上的不同位点结合，并且三联蛋白在确保骨骼肌兴奋 - 收缩偶联期间的快速Ca(2+)释放中起重要作用。
