Calmodulin binding to the 3614-3643 region of RyR1 is not essential for excitation-contraction coupling in skeletal myotubes.
Calmodulin is a ubiquitous Ca(2+) binding protein that modulates the in vitro activity of the skeletal muscle ryanodine receptor (RyR1). Residues 3614-3643 of RyR1 comprise the CaM binding domain and mutations within this region result in a loss of both high-affinity Ca(2+)-bound calmodulin (CaCaM) and Ca(2+)-free CaM (apoCaM) binding (L3624D) or only CaCaM binding (W3620A). To investigate the functional role of CaM binding to this region of RyR1 in intact skeletal muscle, we compared the ability of RyR1, L3624D, and W3620A to restore excitation-contraction (EC) coupling after expression in RyR1-deficient (dyspedic) myotubes. W3620A-expressing cells responded normally to 10 mM caffeine and 500 microM 4-chloro-m-cresol (4-cmc). Interestingly, L3624D-expressing cells displayed a bimodal response to caffeine, with a large proportion of cells ( approximately 44%) showing a greatly attenuated response to caffeine. However, high and low caffeine-responsive L3624D-expressing myotubes exhibited Ca(2+) transients of similar magnitude after activation by 4-cmc (500 microM) and electrical stimulation. Expression of either L3624D or W3620A in dyspedic myotubes restored both L-type Ca(2+) currents (retrograde coupling) and voltage-gated SR Ca(2+) release (orthograde coupling) to a similar degree as that observed for wild-type RyR1, although L-current density was somewhat larger and activated at more hyperpolarized potentials in W3620A-expressing myotubes. The results indicate that CaM binding to the 3614-3643 region of RyR1 is not essential for voltage sensor activation of RyR1.
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Kristen M S
KM
O'Connell
Naohiro
N
Yamaguchi
Gerhard
G
Meissner
Robert T
RT
Dirksen
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine