Allosterically Coupled Calcium and Magnesium Binding Sites are Unmasked by Ryanodine Receptor Chimeras

摘要

We studied cation regulation of wild type ryanodine receptor type 1 (WTRyR1), type 3 (WTRyR3) and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [³H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca²⁺ titrations with WTRyR3 and three chimeras show biphasic activation that is allosterically coupled to attenuated inhibition relative to WTRyR1. Chimeras show biphasic Mg²⁺ inhibition profiles at 3 and 10μM Ca²⁺, no observable inhibition at 20μM Ca²⁺ and monophasic inhibition at 100μM Ca²⁺. Ca²⁺ imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca²⁺ transients with inhibition kinetics that are significantly slower than those expressing WTRyR1 or WTRyR3. Four new aspects of RyR regulation are evident: 1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, 2) Ca²⁺ facilitates removal of the inherent Mg²⁺ block, 3) WTRyR3 exhibits reduced cooperativity between H activation sites when compared to WTRyR1, and 4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca transients.