Luminal Ca2+ regulation of RyR1 Ca2+ channel leak activation and inactivation in sarcoplasmic reticulum membrane vesicles

摘要

In this study, we tested the hypothesis that the RyR1 Ca~(2+) channel closure is sensitive to outward trans-SR membrane Ca~(2+) gradients established by SERCA1 pumping. To perform these studies, we employed stopped-flow rapid-kinetic fluorescence methods to measure and assess how variation in trans-SR membrane Ca~(2+) distribution affects evolution of RyR1 Ca~(2+) leaks in RyR1/ CASQ1/SERCA1-rich membrane vesicles. Our studies showed that rapid filling of a Mag-Fura-2-sensitive free Ca~(2+) pool during SERCA1-mediated Ca~(2+) sequestration appears to be a crucial condition allowing RyR1 Ca~(2+) channels to close once reloading of luminal Ca~(2+) stores is complete. Disruption in the filling of this pool caused activation of Ruthenium Red inhibitable RyR1 Ca~(2+) leaks, suggesting that SERCA1 pump formation of outward Ca~(2+) gradients is an important aspect of Ca~(2+) flux control channel opening and closing. In addition, our observed ryanodine-induced shift in luminal Ca~(2+) from free to a CTC–Ca~(+)-sensitive, CASQ1-associated bound compartment underscores the complex organization and regulation of SR luminal Ca~(2+). Our study provides strong evidence that RyR1 functional states directly and indirectly influence the compartmentation of luminal Ca~(2+). This, in turn, is influenced by the activity of SERCA1 pumps to fill luminal pools while synchronously reducing Ca~(2+) levels on the cytosolic face of RyR1 channels.