Functional analysis of ryanodine receptor using molecular genetic technique

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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.