Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation. II. Resolution of a caffeine-ryanodine paradox

Our previous study demonstrated a disparity of action between two establish ed pharmacological modulators of the same calcium (Ca2+) release channel, t he ryanodine receptor (RyR). Specifically, we observed that caffeine sensit ivity was elicited at earlier stages of development than that of ryanodine. In the present study, we offer a hypothesis to resolve this paradox. We pr ovide evidence that ryanodine acts as a pure uncompetitive inhibitor of Ca2 + transport, with respect to Ca2+ itself, This explains why little ryanodin e inhibition was observed at low Ca2+ concentrations, while maximal ryanodi ne inhibition was observed at saturating Ca2+ concentrations. In order to e xclude the possibility of nonspecific ryanodine actions as an alternative e xplanation, we established the phenomenon of capacitative calcium entry (CC E) for L6 cells, Since it is known that CCE is inversely correlated with [C a2+] of the ER/SR lumen, the extent of CCE is therefore an indirect measure of Ca2+ concentration within the SR. We also demonstrated the functional p athway for Ca2+ entry. Employing pharmacological inhibitors, we found that a T-type plasma membrane channel was predominant in the myoblasts, while an L-type channel was predominant in the adult myotubes. Our data using these inhibitors made nonspecific ryanodine actions an unlikely explanation of t he disparity in action between ryanodine and caffeine. Moreover, we found n o evidence that inositol trisphosphate, a proposed regulator of CCE for oth er cells, could influence CCE in L6 cells. We conclude that the disparity b etween caffeine and ryanodine can be explained by Ca2+ dependence of ryanod ine action. This study may also offer an explanation of other studies showi ng unclear actions of ryanodine binding and action.