Changes in Ryanodine Receptor-Mediated Calcium Release During Skeletal Muscle Differentiation. II. Resolution of a Caffeine-Ryanodine Paradox

Abstract

Our previous study demonstrated a disparity of action between two established pharmacological modulators of the same calcium (Ca²⁺) release channel, the ryanodine receptor (RyR). Specifically, we observed that caffeine sensitivity was elicited at earlier stages of development than that of ryanodine. In the present study, we offer a hypothesis to resolve this paradox. We provide evidence that ryanodine acts as a pure uncompetitive inhibitor of Ca²⁺ transport, with respect to Ca²⁺ itself. This explains why little ryanodine inhibition was observed at low Ca²⁺ concentrations, while maximal ryanodine inhibition was observed at saturating Ca²⁺ concentrations. In order to exclude the possibility of nonspecific ryanodine actions as an alternative explanation, we established the phenomenon of capacitative calcium entry (CCE) for L6 cells. Since it is known that CCE is inversely correlated with [Ca²⁺] 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 pathway 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 the disparity in action between ryanodine and caffeine. Moreover, we found no evidence that inositol trisphosphate, a proposed regulator of CCE for other cells, could influence CCE in L6 cells. We conclude that the disparity between caffeine and ryanodine can be explained by Ca²⁺ dependence of ryanodine action. This study may also offer an explanation of other studies showing unclear actions of ryanodine binding and action.