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

In this study, we tested the hypothesis that the RyR1 Ca2+ channel closure is sensitive to outward trans-SR membrane Ca2+ 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 Ca2+ distribution affects evolution of RyR1 Ca2+ leaks in RyR1/ CASQ1/SERCA1-rich membrane vesicles. Our studies showed that rapid filling of a Mag-Fura-2-sensitive free Ca2+ pool during SERCA1-mediated Ca2+ sequestration appears to be a crucial condition allowing RyR1 Ca2+ channels to close once reloading of luminal Ca2+ stores is complete. Disruption in the filling of this pool caused activation of Ruthenium Red inhibitable RyR1 Ca2+ leaks, suggesting that SERCA1 pump formation of outward Ca2+ gradients is an important aspect of Ca2+ flux control channel opening and closing. In addition, our observed ryanodine-induced shift in luminal Ca2+ from free to a CTC-Ca+-sensitive, CASQ1-associated bound compartment underscores the complex organization and regulation of SR luminal Ca2+. Our study provides strong evidence that RyR1 functional states directly and indirectly influence the compartmentation of luminal Ca2+. This, in turn, is influenced by the activity of SERCA1 pumps to fill luminal pools while synchronously reducing Ca2+ levels on the cytosolic face of RyR1 channels.

Spectroscopic determination of sarcoplasmic reticulum Ca2+ uptake and Ca2+ release.

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.