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Ca²⁺ Stores Regulate Ryanodine Receptor Ca²⁺ Release Channels via Luminal and Cytosolic Ca²⁺ Sites

School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales, Australia

Correspondence: Address reprint requests to Dr. Derek Laver, Tel.: 61-2-4921-8732; E-mail: derek.laver{at}newcastle.edu.au.

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, 60 µM affinity), a cytoplasmic activation site (A-site, 0.9 µM affinity), and a novel cytoplasmic inactivation site (I₂-site, 1.2 µM 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 (1 ms duration at 1–10 s^–1) 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.

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