title: Ca2+ activation of RyR1 is not necessary for the initiation of skeletal-type excitation-contraction coupling.
abstract: Although an elevation in myoplasmic Ca2+ can activate the skeletal muscle ryanodine receptor (RyR1), the function of this Ca2+ activation is unclear because extracellular Ca2+ influx is unnecessary for skeletal-type EC coupling. To determine whether Ca2+ 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 Ca2+. Proc. Natl. Acad. Sci. USA. 98:2865-2870). Analysis after reconstitution into planar lipid bilayers revealed that E4032A-RyR1 was negligibly activated by 100 microM Ca2+ (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 microM free Ca2+ to 0.005 in 2 mM free Ca2+). Thus, the E4032A mutation caused a nearly complete suppression of activation of RyR1 by Ca2+. Depolarization of E4032A-RyR1-expressing myotubes elicited L-type Ca2+ currents of approximately normal size and myoplasmic Ca2+ transients that were skeletal-type, but about fivefold smaller than those for wild-type RyR1. The reduced amplitude of the Ca2+ transient is consistent either with the possibility that Ca2+ activation amplifies Ca2+ release during EC coupling, or that the E4032A mutation generally inhibits activation of RyR1. In either case, Ca2+ activation of RyR1 does not appear to be necessary for the initiation of Ca2+ release during EC coupling in skeletal muscle.
authors.affiliation: Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523, USA.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: O'Brien
authors.forename: Wei
authors.initials: W
authors.lastname: Feng
authors.forename: Paul D
authors.initials: PD
authors.lastname: Allen
authors.forename: S R Wayne
authors.initials: SR
authors.lastname: Chen
authors.forename: Isaac N
authors.initials: IN
authors.lastname: Pessah
authors.forename: Kurt G
authors.initials: KG
authors.lastname: Beam
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D018076:DNA, Complementary; D008051:Lipid Bilayers / Q000737:chemistry; D008322:Mammals; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000235:genetics / Q000502:physiology*
chemical_list: D018076:DNA, Complementary; D008051:Lipid Bilayers; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D000255:Adenosine Triphosphate; D002118:Calcium