Interdependence of ryanodine binding, oligomeric receptor interactions, and Ca 2+ release regulation in junctional sarcoplasmic reticulum

Abstract

We have examined ryanodine binding to its receptor (RR) and compared its effect on Ca 2+ release to the Ca 2+ release triggered by Ca 2+ plus ATP, using vesicular fragments of junctional terminal cisternae (JTC) obtained from skeletal muscle. Ryanodine binding is slow (taking hours or days to complete) and is highly temperature ( Q 10 = 4) and Ca 2+ dependent. At equilibrium, the extent of binding increases as the concentration of ryanodine is raised above 10 −9 m, exhibiting negative cooperativity and reaching the stoichiometry of the 560,000-Da RR chains near 10 −5 m ryanodine. The specificity of the high affinity binding is demonstrated by competitive binding of ryanodine analogs. Kinetic studies using rapid filtration show that, in the absence of ryanodine, rapid ( k = 15 s −1) release of Ca 2+ follows a triggering exposure of loaded JTC vesicles to perfusion media containing Ca 2+ plus ATP. Induction of this release has no lag period and displays minimal temperature dependence. In contrast, prolonged exposure of JTC vesicles to low (10 −7 m) ryanodine concentrations changes the JTC to a state permitting slow ( k = 1 s −1) release of Ca 2+ even in the absence of the Ca 2+ plus ATP trigger. Higher (>μ m) concentrations of ryanodine do not allow any Ca 2+ release and prevent even the release normally triggered by Ca 2+ plus ATP. Our data suggest that ryanodine binds to the open state of the tetrameric RR, inducing protein conformational changes and altered oligomeric interactions. Binding of the first molecule of ryanodine to one of the four binding sites on the receptor produces a partially closed and low conductance state of the Ca 2+ release channel and reduces the ryanodine binding affinity of the remaining sites. Ryanodine occupancy of all four binding sites on the receptor completes closure of the Ca 2+ channel and blocks the triggering action of Ca 2+ plus ATP. The tetrameric association of the RR chains is demonstrated by crosslinking with bifunctional reagents, generating crosslinked tetramers that retain ryanodine binding and Ca 2+ release functions.