Luminal Ca²⁺–regulated Mg²⁺ Inhibition of Skeletal RyRs Reconstituted as Isolated Channels or Coupled Clusters

In resting muscle, cytoplasmic Mg²⁺ is a potent inhibitor of Ca²⁺ release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca²⁺ sites (A-sites) thus preventing Ca²⁺ activation. We investigate the effects of luminal and cytoplasmic Ca²⁺ on Mg²⁺ inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg²⁺ inhibits RyRs at the A-site in the absence of Ca²⁺, indicating that Mg²⁺ is an antagonist and does not simply prevent Ca²⁺ activation. Cytoplasmic Ca²⁺ and Cs⁺ decreased Mg²⁺ affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca²⁺ regulation of Ca²⁺ release whereby increasing luminal [Ca²⁺] decreases the A-site affinity for cytoplasmic Mg²⁺ by a noncompetitive, allosteric mechanism that is independent of Ca²⁺ flow. Ryanodine increases the Ca²⁺ sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca²⁺, indicating that ryanodine activates independently of Ca²⁺. We describe a model for ion binding at the A-sites that predicts that modulation of Mg²⁺ inhibition by luminal Ca²⁺ is a significant regulator of Ca²⁺ release from the SR. We detected coupled gating of RyRs due to luminal Ca²⁺ permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca²⁺ and cytoplasmic Mg²⁺ did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca²⁺ was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively “immune” to Ca²⁺ emanating from their own pore but sensitive to Ca²⁺ from neighboring channels.