Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras.

ABSTRACT

We studied cation regulation of wild-type ryanodine receptor type 1 ((WT)RyR1), type 3 ((WT)RyR3), and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [(3)H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca(2+) titrations with (WT)RyR3 and three chimeras show biphasic activation that is allosterically coupled to an attenuated inhibition relative to (WT)RyR1. Chimeras show biphasic Mg(2+) inhibition profiles at 3 and 10 microM Ca(2+), no observable inhibition at 20 microM Ca(2+) and monophasic inhibition at 100 microM Ca(2+). Ca(2+) imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca(2+) transients with inhibition kinetics that are significantly slower than those expressing (WT)RyR1 or (WT)RyR3. Four new aspects of RyR regulation are evident (1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, (2) Ca(2+) facilitates removal of the inherent Mg(2+) block, (3) (WT)RyR3 exhibits reduced cooperativity between H activation sites when compared to (WT)RyR1, and (4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca(2+) transients.