Functional analysis of ryanodine receptor using molecular genetic technique

利用基因敲除等分子遗传学方法研究了Ryanodine受体亚型（RyR-1、RyR-2和RyR-3）的功能。我们在这项研究项目中的主要发现如下。1.缺乏RyR-3的突变小鼠的产生和表征。由于RyR-3的生理作用是完全未知的，我们已经通过产生缺乏RyR-3的突变小鼠来研究其作用。突变小鼠的分析结果显示RyR-3在肌肉细胞和淋巴细胞中的非必需功能。然而，RyR-3的基本功能已经在某些类型的神经元中被提出。2.二氢吡啶受体（DHPR）在缺乏RyR-1的突变肌肉中的功能。DHPR和RyR-1之间的直接相互作用已被提出构成骨骼肌细胞中的兴奋-收缩（e-c）偶联。我们已经分析了钙电流通过DHPR在突变体肌肉缺乏RyR-1，并发现钙电流的显着减少和缺乏其促进突变体细胞。结果支持DHPR和RyR-1.3之间直接相互作用的想法。骨骼肌兰尼碱受体对电-电偶联的亚型特异性。我们已经表明，骨骼肌含有RyR-1和RyR-3，RyR-1的缺失导致e-c解偶联。接着，将RyR-2 cDNA导入突变细胞中。RyR-2的表达并没有恢复e-c解偶联，但诱导自发钙振荡。结果表明，RyR-1的能力和RyR-2和3不能促进骨骼肌中的钙信号传导。

The functions of ryanodine receptor subtypes (RyR-1,2 and 3) have been investigated by means of molecular genetic methords, for example generation of knockout moce. Our major findings in this research project are listed below. 1. Generation and characterization of mutant mice lacking RyR-3. Because the physiological role of RyR-3 was totally unknown, we have investigated the role by the generation of mutant mice lacking RyR-3. The results of analysis of the mutant mice showed non-essential function of RyR-3 in muscle cells and lymphocytes. However, the essential function of RyR-3 has been suggested in certain types of neurons. 2. Function of the dihydropyridine receptor (DHPR) in mutant muscle lacking RyR-1. Direct interaction between DHPR and RyR-1 has been proposed to constitute excitation-contraction (e-c) coupling in skeletal muscle cells. We have analyed calcium currents via DHPR in the mutant muscles lacking RyR-1, and have found significant decrease of calcium currents and absence of its facilitation in the mutant cells. The results suport the idea of the direct interaction between DHPR and RyR-1.3. Subtype specificity of ryanodine receptor for e-c coupling in skeletal muscle. We have shown that skeletal muscle contains RyR-1 and RyR-3, and loss of RyR-1 results in e-c uncoupling. Next, RyR-2 cDNA was introduced in the mutant cells. The RyR-2 expression did not restore e-c uncoupling but induced spontaneous calcium ocillasion. The results indicated ability of RyR-1 and inability of RyR-2 and 3 to contribute for calcium signalling in skeletal muscle.