Expression of Ca²⁺-Induced Ca²⁺ Release Channel Activity from Cardiac Ryanodine Receptor cDNA in Chinese Hamster Ovary Cells

抄録

We constructed an expression plasmid (pMAMCRR51) that carried the entire proteincoding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthineguanine phosphoribosyltransferase (gpt). Chinese hamster ovary (CHO) cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca²⁺ transients were selected. Immunoprecipitation with a monoclonal antibody against the canine cardiac ryanodine receptor revealed that the cell clones thus selected exhibited Ca²⁺-dependent [³H]ryanodine binding activity, which was stimulated by 5mM ATP or 1M KCl. The apparent dissociation constant (K_d) for [³H] ryanodine was 6.6nM in 1M KCl, which was similar to the K_d obtained with cardiac microsomes. Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in M_r from the ryanodine receptor in canine cardiac microsomes. The ryanodine binding activity expressed in CHO cells increased significantly after dexamethasone induction. In saponin-skinned CHO cells transfected with pMAMCRR51, micromolar Ca²⁺ or millimolar caffeine evoked rapid Ca²⁺ release from the intracellular Ca²⁺ stores. In skinned control CHO cells, we did not observe such Ca²⁺ release activity. These results clearly demonstrate that the cardiac ryanodine receptor is stably expressed in internal membranes of CHO cells and functions as Ca²⁺-induced Cal²⁺ release channels.