Ca super(2+) Stores Regulate Ryanodine Receptor Ca super(2+) Release Channels via Luminal and Cytosolic Ca super(2+) Sites

The free [Ca super(2+)] in endoplasmic/sarcoplasmic reticulum Ca super(2+) stores regulates excitability of Ca super(2+) release by stimulating the Ca super(2+) release channels. Just how the stored Ca super(2+) regulates activation of these channels is still disputed. One proposal attributes luminal Ca super(2+)-activation to luminal facing regulatory sites, whereas another envisages Ca super(2+) permeation to cytoplasmic sites. This study develops a unified model for luminal Ca super(2+) activation for single cardiac ryanodine receptors (RyR sub(2)) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR sub(2) involves three modes of action associated with Ca super(2+) sensors in different parts of the molecule; a luminal activation site (L-site, 60 mu M affinity), a cytoplasmic activation site (A-site, 0.9 mu M affinity), and a novel cytoplasmic inactivation site (I sub(2)-site, 1.2 mu M affinity). RyR activation by luminal Ca super(2+)is demonstrated to occur by amultistep process dubbed luminal-triggered Ca super(2+) feedthrough. Ca super(2+) binding to the Z-site initiates brief openings (1 ms duration at 1-10 s super(-1)) allowing luminal Ca super(2+) to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR sub(2) mutations on a range of Ca super(2+)-mediated physiological and pathological processes.