Expression of Ca(2+)-induced Ca2+ release channel activity from cardiac ryanodine receptor cDNA in Chinese hamster ovary cells.
We constructed an expression plasmid (pMAMCRR51) that carried the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthine-guanine 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 Ca(2+)-dependent [3H]ryanodine binding activity, which was stimulated by 5 mM ATP or 1 M KCl. The apparent dissociation constant (Kd) for [3H]ryanodine 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 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 Ca(2+)-induced Ca2+ release channels.
Imagawa
T
T
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka.
Nakai
J
J
Takeshima
H
H
Nakasaki
Y
Y
Shigekawa
M
M
D000818:Animals; D016466:CHO Cells / Q000378:metabolism / Q000502:physiology*; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D015220:Calcium Channels / Q000502:physiology*; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D007425:Intracellular Membranes / Q000502:physiology / Q000648:ultrastructure; D009206:Myocardium / Q000648:ultrastructure*; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000235:genetics* / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel; D012503:Saponins / Q000494:pharmacology; D014162:Transfection
D015220:Calcium Channels; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012503:Saponins; D002110:Caffeine; D003907:Dexamethasone; D004247:DNA; D002118:Calcium