We constructed an expression plasmid (pMAMCRR51) that carried the entire proteincoding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexameth-asone-inducible mouse mammary tumor virus promoter and Escherichia ccli 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] binding activity, which was stimulated by 5 mM ATP or 1 M KCl. The apparent dissociation constant (Kd) for [3H] was 6.6 nM in 1 M KCl, which was similar to the Kd obtained with cardiac microsomes. Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in Mr 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 Ca2+ release channels.
|ジャーナル||Journal of biochemistry|
|出版ステータス||Published - 1992 10|
ASJC Scopus subject areas
- Molecular Biology