Ca 2+ Activation of RyR1 Is Not Necessary for the Initiation of Skeletal-Type Excitation-Contraction Coupling

Abstract

Although an elevation in myoplasmic Ca 2+ can activate the skeletal muscle ryanodine receptor (RyR1), the function of this Ca 2+ activation is unclear because extracellular Ca 2+ influx is unnecessary for skeletal-type EC coupling. To determine whether Ca 2+ activation of RyR1 is necessary for the initiation of skeletal-type EC coupling, we examined the behavior of RyR1 with glutamate 4032 mutated to alanine (E4032A-RyR1) because this mutation had been shown to dramatically reduce activation by Ca 2+. Proc. Natl. Acad. Sci. USA. 98:2865–2870). Analysis after reconstitution into planar lipid bilayers revealed that E4032A-RyR1 was negligibly activated by 100 μM Ca 2+ ( P o too low to be measured). Even in the presence of both 2 mM caffeine and 2 mM ATP, P o remained low for E4032A-RyR1 (ranging from <0.0001 in 100 μM free Ca 2+ to 0.005 in 2 mM free Ca 2+). Thus, the E4032A mutation caused a nearly complete suppression of activation of RyR1 by Ca 2+. Depolarization of E4032A-RyR1-expressing myotubes elicited L-type Ca 2+ currents of approximately normal size and myoplasmic Ca 2+ transients that were skeletal-type, but about fivefold smaller than those for wild-type RyR1. The reduced amplitude of the Ca 2+ transient is consistent either with the possibility that Ca 2+ activation amplifies Ca 2+ release during EC coupling, or that the E4032A mutation generally inhibits activation of RyR1. In either case, Ca 2+ activation of RyR1 does not appear to be necessary for the initiation of Ca 2+ release during EC coupling in skeletal muscle.