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

摘要

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 (Po) of ryanodine-modified channels was only marginally altered at pCa 10 (pCa = −log10 Ca concentration). However, at −30 mV, the Po 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 Po of ryanodine-modified channels at low Ca²⁺ concentrations at both −30 and +30 mV. Caffeine, ATP, and ruthenium red were modulators of the Po of ryanodine-modified channels. In a [³H]ryanodine binding assay, [³H]ryanodine dissociation from the high-affinity binding site was found to be Ca²⁺ sensitive, with an IC50 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.