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 [H-3]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca2+ 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 Mg2+ inhibition profiles at 3 and 10 mu M Ca2+, no observable inhibition at 20 mu M Ca2+ and monophasic inhibition at 100 mu M Ca2+. Ca2+ imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca2+ 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) Ca2+ facilitates removal of the inherent Mg2+ 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 Ca2+ transients. (c) 2007 Elsevier Inc. All rights reserved.