title: Ryanodine sensitizes the cardiac Ca(2+) release channel (ryanodine receptor isoform 2) to Ca(2+) activation and dissociates as the channel is closed by Ca(2+) depletion.
abstract: In single-channel recordings, the rabbit cardiac Ca(2+) 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 Ca(2+) added to the cis (cytoplasmic) side and, at pCa 10, was reduced to less than 0.27. The EC(50) value for channel opening was about pCa 8. No significant Ca(2+) inactivation was observed for ryanodine-modified channels at either -30 mV or +30 mV. The opening of unmodified Ca(2+) channels is Ca(2+) sensitive, with an EC(50) value of about pCa 6 (two orders of magnitude less sensitive than ryanodine-modified channels) and IC(50) values of pCa 2.2 at -30 mV and 2.5 at +30 mV. Mg(2+) decreased the P(o) of ryanodine-modified channels at low Ca(2+) concentrations at both -30 and +30 mV. Caffeine, ATP, and ruthenium red were modulators of the P(o) of ryanodine-modified channels. In a [(3)H]ryanodine binding assay, [(3)H]ryanodine dissociation from the high-affinity binding site was found to be Ca(2+) sensitive, with an IC(50) of pCa 7.1. High concentrations of unlabeled ryanodine prevented [(3)H]ryanodine dissociation, but ruthenium red accelerated dissociation. These results suggest that ryanodine sensitizes Ca(2+) activation of the Ca(2+) release channel and desensitizes Ca(2+) inactivation through an allosteric interaction. [(3)H]Ryanodine dissociates from the high-affinity site when the channel is closed by removal of Ca(2+), implying that high-affinity ryanodine and Ca(2+) binding sites are linked through either short- or long-range interactions, probably involving conformational changes.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada M5G 1L6.
authors.forename: G G
authors.initials: GG
authors.lastname: Du
authors.forename: X
authors.initials: X
authors.lastname: Guo
authors.forename: V K
authors.initials: VK
authors.lastname: Khanna
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism* / Q000502:physiology; D004594:Electrophysiology; D015640:Ion Channel Gating; D009206:Myocardium / Q000378:metabolism; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism* / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism* / Q000502:physiology
chemical_list: D015220:Calcium Channels; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002118:Calcium