Binding property of avian skeletal muscle ryanodine receptor isoforms with dihydropyridine receptor and calmodulin

摘要

Ca2+ release during excitation–contraction coupling in avian skeletal muscle is controlled by two ryanodine receptor isoforms, αRYR and βRYR. Two other proteins, dihydropyridine receptor (DHPR) and calmodulin (CaM), have been shown to play important roles in regulating the RYR channel activity. In the current study, we measured the protein contents of DHPR and RYR in turkey skeletal muscle and obtained a ratio of 1:1 between DHPR and αRYR which suggests that only a subpopulation of αRYR is interacting with DHPR. Two CaM derivatives, the photoactivable crosslinking probe [125I]-Bz-CaM and metabolically labeled probe [35S]CaM, were used to study the interaction between CaM and RYR isoforms in turkey skeletal muscle. The αRYR and βRYR displayed a marked difference in their CaM binding behavior. At a Ca2+ concentration of 200 μM, CaM bound to both isoforms at a ratio of one CaM molecule per one RYR subunit. At a Ca2+ concentration of <10 nM, CaM bound primarily to αRYR and the binding affinity was significantly lower than that at micromolar level of Ca2+ concentration. Cloning and sequencing of putative CaM binding sites in αRYR and βRYR suggests that differences in primary structures of the CaM binding sites of each RYR isoform may contribute to the differential CaM binding behavior of αRYR and βRYR.