Ca²⁺ stores regulate ryanodine receptor Ca²⁺ release channels via luminal and cytosolic Ca²⁺ sites

The free [Ca²⁺] in endoplasmic/sarcoplasmic reticulum Ca²⁺ stores regulates excitability of Ca²⁺ release by stimulating the Ca²⁺ release channels. Just how the stored Ca²⁺ regulates activation of these channels is still disputed. One proposal attributes luminal Ca²⁺-activation to luminal facing regulatory sites, whereas another envisages Ca²⁺ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca²⁺ activation for single cardiac ryanodine receptors (RyR2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca²⁺ sensors in different parts of the molecule; a luminal activation site (L-site, 60M affinity), a cytoplasmic activation site (A-site, 0.9M affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2M affinity). RyR activation by luminal Ca²⁺ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca²⁺ feedthrough. Ca²⁺ binding to the L-site initiates brief openings (1ms duration at 110s1) allowing luminal Ca²⁺ 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 RyR2 mutations on a range of Ca²⁺-mediated physiological and pathological processes.