Ryanodine sensitizes the cardiac Ca2+ release channel (ryanodine receptor isoform 2) to Ca2+ activation and dissociates as the channel is closed by Ca2+ depletion

In single-channel recordings, the rabbit cardiac Ca2+ release channel (RyR2) is converted to a fully open subconductance state with about 50% of full conductance by micromolar concentrations of ryanodine. At +30 mV, corresponding to a luminal to cytoplasmic cation current, the probability of opening (P-o) of ryanodine-modified channels was only marginally altered at pCa 10 (pCa = -log(10) Ca concentration). However, at -30 mV, the P-o was highly sensitive to Ca2+ added to the cis (cytoplasmic) side and, at pCa 10, was reduced to less than 0.27. The EC50 value for channel opening was about pCa 8. No significant Ca2+ inactivation was observed for ryanodine-modified channels at either -30 mV or +30 mV. The opening of unmodified Ca2+ channels is Ca2+ sensitive, with an EC50 value of about pCa. 6 (two orders of magnitude less sensitive than ryanodine-modified channels) and IC50 values of pCa 2.2 at -30 mV and 2.5 at +30 mV. Mg2+ decreased the P-o of ryanodine-modified channels at low Ca2+ concentrations at both -30 and +30 mV. Caffeine, ATP, and ruthenium red were modulators of the P. of ryanodine-modified channels. In a [H-3]ryanodine binding assay, [3H]ryanodine dissociation from the high-affinity binding site was found to be Ca2+ sensitive, with an I(C5)0 of pCa 7.1. High concentrations of unlabeled ryanodine prevented [3H]ryanodine dissociation, but ruthenium red accelerated dissociation. These results suggest that ryanodine sensitizes Ca2+ activation of the Ca2+ release channel and desensitizes Ca2+ inactivation through an allosteric interaction. [3H]Ryanodine dissociates from the high-affinity site when the channel is closed by removal of Ca2+, implying that high-affinity ryanodine and Ca2+ binding sites are linked through either short- or long-range interactions, probably involving conformational changes.