Gomez Angela C, Holford Timothy W, Yamaguchi Naohiro
Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina; and.
Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, South Carolina.
Am J Physiol Cell Physiol. 2016 Nov 1;311(5):C749-C757. doi: 10.1152/ajpcell.00134.2016. Epub 2016 Aug 24.
Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca and inactivated by higher (∼1 mM) Ca To gain insight into a mechanism underlying Ca-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca-dependent channel inactivation. Activities of these mutant channels were suppressed at 10-100 μM Ca, and the suppressions were relieved by 1 mM Mg The Ca- and Mg-dependent regulation of S2-S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild-type RyR1. Two mutations (T4825I and H4832Y) in the S4-S5 cytoplasmic loop increased Ca affinities for channel activation and decreased Ca affinities for inactivation, but impairment of Ca-dependent inactivation was not as prominent as those of S2-S3 loop mutants. Three mutations (T4082M, S4113L, and N4120Y) in the EF-hand domain showed essentially the same Ca-dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca and Mg Four malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop conferred RyR2-type Ca- and Mg-dependent channel regulation.
骨骼肌兰尼碱受体（RyR1）的通道活性由微摩尔浓度的钙离子激活，并在较高浓度（约1 mM）的钙离子作用下失活。为深入了解RyR1钙离子依赖性失活的机制及其与骨骼肌疾病的关系，我们构建了九个携带恶性高热或中央核肌病相关突变的重组RyR1突变体，并通过[H]兰尼碱结合试验测定了RyR1通道活性。这些突变位于负责RyR1钙离子依赖性失活的RyR1结构域内或附近。位于S2和S3跨膜片段之间的胞质环（S2-S3环）中的四个RyR1突变（F4732D、G4733E、R4736W和R4736Q）极大地降低了钙离子依赖性通道失活。这些突变通道的活性在10-100 μM钙离子浓度下受到抑制，而1 mM镁离子可缓解这种抑制作用。S2-S3环RyR1突变体的钙离子和镁离子依赖性调节与心脏型RyR（RyR2）相似，而非野生型RyR1。S4-S5胞质环中的两个突变（T4825I和H4832Y）增加了通道激活的钙离子亲和力，降低了失活的钙离子亲和力，但钙离子依赖性失活的损害不如S2-S3环突变体明显。EF手结构域中的三个突变（T4082M、S4113L和N4120Y）表现出与野生型RyR1基本相同的钙离子依赖性通道调节。结果表明，九个与骨骼肌疾病相关的RyR1突变体对钙离子和镁离子的调节方式不同。S2-S3环中的四个与恶性高热相关的RyR1突变赋予了RyR2型钙离子和镁离子依赖性通道调节。
