Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras.
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.
Voss
Andrew A
AA
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
Allen
Paul D
PD
Pessah
Isaac N
IN
Perez
Claudio F
CF
D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D008274:Magnesium; D002118:Calcium