Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy T V, MacLennan D H
Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Toronto, Ontario M5G 1L6, Canada.
J Biol Chem. 1997 Oct 17;272(42):26332-9. doi: 10.1074/jbc.272.42.26332.
Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle in which a potentially fatal hypermetabolic crisis can be triggered by commonly used anesthetic agents. To date, 17 mutations in the human RYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been associated with MH and/or CCD. Although many of these mutations have been linked to MH and/or CCD, with high lod (log of the odds favoring linkage versus nonlinkage) scores, others have been found in single, small families. Independent biochemical evidence for a causal role for these mutations in MH is available for only two mutants. Mutations corresponding to the human MH mutations were made in a full-length rabbit RYR1 cDNA, and wild type and mutant cDNAs were transfected into HEK-293 cells. After about 48 h, intact cells were loaded with the fluorescent Ca2+ indicator, fura-2, and intracellular Ca2+ release, induced by caffeine or halothane, was measured by photometry. Ca2+ release in cells expressing MH or CCD mutant ryanodine receptors was invariably significantly more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild type receptors or receptors mutated in other regions of the molecule. Linear regression analysis showed that there is a strong correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants measured by the cellular Ca2+ photometry assay and by the clinical in vitro caffeine halothane contracture test (IVCT). The correlation was weaker, however, for halothane (r = 0.49, p > 0.05). Abnormal sensitivity in the Ca2+ photometry assay provides supporting evidence for a causal role in MH for each of 15 single amino acid mutations in the ryanodine receptor. The study demonstrates the usefulness of the cellular Ca2+ photometry assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants.
恶性高热（MH）和中央轴空病（CCD）是骨骼肌的常染色体显性疾病，常用麻醉剂可引发潜在致命的代谢亢进危机。迄今为止，编码骨骼肌肌浆网Ca2+释放通道（雷诺丁受体）的人类RYR1基因中的17种突变已与MH和/或CCD相关联。尽管这些突变中有许多已与MH和/或CCD相关联，连锁对数（支持连锁与非连锁的对数）得分很高，但其他突变仅在单个小家族中被发现。只有两个突变体有关于这些突变在MH中起因果作用的独立生化证据。在全长兔RYR1 cDNA中产生了与人类MH突变相对应的突变，并将野生型和突变型cDNA转染到HEK-293细胞中。约48小时后，完整细胞用荧光Ca2+指示剂fura-2加载，通过光度法测量由咖啡因或氟烷诱导的细胞内Ca2+释放。与表达野生型受体或分子其他区域突变受体的细胞中的Ca2+释放相比，表达MH或CCD突变雷诺丁受体的细胞中的Ca2+释放对低浓度咖啡因和氟烷总是明显更敏感。线性回归分析表明，通过细胞Ca2+光度法测定和临床体外咖啡因氟烷挛缩试验（IVCT）测定的不同RYR1突变体的咖啡因敏感性之间存在强相关性（r = 0.95，p < 0.001）。然而，对于氟烷，相关性较弱（r = 0.49，p > 0.05）。Ca2+光度法测定中的异常敏感性为雷诺丁受体中的15个单氨基酸突变中的每一个在MH中起因果作用提供了支持证据。该研究证明了细胞Ca2+光度法在评估特定雷诺丁受体突变体对咖啡因和氟烷敏感性方面的有用性。
