Selectively suppressed Ca2+-induced Ca2+ release activity of alpha-ryanodine receptor (alpha-RyR) in frog skeletal muscle sarcoplasmic reticulum: potential distinct modes in Ca2+ release between alpha- and beta-RyR.
We reported earlier that the two ryanodine receptor (RyR) isoforms (alpha- and beta-RyR) purified from frog skeletal muscle were equipotent in the Ca(2+)-induced Ca(2+) release (CICR) activity (Murayama, T., Kurebayashi, N., and Ogawa, Y. (2000) Biophys. J. 78, 1810-1824). Whether this is also the case with the native Ca(2+) release channel in the sarcoplasmic reticulum (SR), however, remains to be determined. Taking advantage of the facts that [(3)H]ryanodine binds only to the open form of the channels and that it is practically irreversible at 4 degrees C, we devised a method to separate the total binding to contributions of alpha- and beta-RyR, using immunoprecipitation with an alpha-RyR-specific monoclonal antibody. Surprisingly, the binding of alpha-RyR was strongly suppressed to as low as approximately 4% that of beta-RyR in the SR vesicles. The two isoforms, however, showed no difference in sensitivity to Ca(2+), adenine nucleotides, or caffeine. This reduced binding of alpha-RyR was ascribed to the low affinity for [(3)H]ryanodine, with no change in the maximal binding sites. Solubilization of SR with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid partly remedied this nonequivalence, whereas 1 m NaCl was ineffective. 12-kDa FK506-binding protein (FKBP12), however, could not be responsible for it, because FK506 treatment did not eliminate the suppression, in contrast to marked removal of 12-kDa FK506-binding protein from alpha-RyR. These results suggest that alpha-RyR in the SR may serve Ca(2+) release in a mode other than CICR, being selectively suppressed in CICR.
Murayama
T
T
Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
Ogawa
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000276:immunology / Q000378:metabolism; D011892:Rana catesbeiana; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000145:classification / Q000276:immunology / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D016559:Tacrolimus / Q000494:pharmacology; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism
D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D022061:Tacrolimus Binding Protein 1A; D002118:Calcium; D016559:Tacrolimus