Luminal Ca2+ Regulation of RyR1 Ca2+ Channel Leak Activation and Inactivation in Sarcoplasmic Reticulum Membrane Vesicles

In this study, we tested the hypothesis that 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 HSR 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 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 both fill luminal pools while synchronously reducing Ca2+ levels on the cytosolic face of RyR1 channels.

The sarcoplasmic Ca2+-ATPase: design of a perfect chemi-osmotic pump

The sarcoplasmic (SERCA 1a) Ca2+-ATPase is a membrane protein abundantly present in skeletal mucles where it functions as an indispensable component of the excitation–contraction coupling, being at the expense of ATP hydrolysis involved in Ca2+/H+ exchange with a high thermodynamic efficiency across the sarcoplasmic reticulum membrane. The transporter serves as a prototype of a whole family of cation transporters, the P-type ATPases, which in addition to Ca2+ transporting proteins count Na+, K+-ATPase and H+, K+-, proton- and heavy metal transporting ATPases as prominent members. The ability in recent years to produce and analyze at atomic (2·3–3 Å) resolution 3D-crystals of Ca2+-transport intermediates of SERCA 1a has meant a breakthrough in our understanding of the structural aspects of the transport mechanism. We describe here the detailed construction of the ATPase in terms of one membraneous and three cytosolic domains held together by a central core that mediates coupling between Ca2+-transport and ATP hydrolysis. During turnover, the pump is present in two different conformational states, E1 and E2, with a preference for the binding of Ca2+ and H+, respectively. We discuss how phosphorylated and non-phosphorylated forms of these conformational states with cytosolic, occluded or luminally exposed cation-binding sites are able to convert the chemical energy derived from ATP hydrolysis into an electrochemical gradient of Ca2+ across the sarcoplasmic reticulum membrane. In conjunction with these basic reactions which serve as a structural framework for the transport function of other P-type ATPases as well, we also review the role of the lipid phase and the regulatory and thermodynamic aspects of the transport mechanism.