title: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.
abstract: In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.
authors.affiliation: Dept. of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Yamaguchi
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Prosser
authors.forename: Farshid
authors.initials: F
authors.lastname: Ghassemi
authors.forename: Le
authors.initials: L
authors.lastname: Xu
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Pasek
authors.forename: Jerry P
authors.initials: JP
authors.lastname: Eu
authors.forename: Erick O
authors.initials: EO
authors.lastname: Hernández-Ochoa
authors.forename: Brian R
authors.initials: BR
authors.lastname: Cannon
authors.forename: Paul T
authors.initials: PT
authors.lastname: Wilder
authors.forename: Richard M
authors.initials: RM
authors.lastname: Lovering
authors.forename: David
authors.initials: D
authors.lastname: Weber
authors.forename: Werner
authors.initials: W
authors.lastname: Melzer
authors.forename: Martin F
authors.initials: MF
authors.lastname: Schneider
authors.forename: Gerhard
authors.initials: G
authors.lastname: Meissner
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism* / Q000502:physiology; D005260:Female; D008297:Male; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology; D053580:Muscle Strength / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009418:S100 Proteins / Q000378:metabolism* / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism* / Q000502:physiology
chemical_list: D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D009418:S100 Proteins; C110294:S100A1 protein; D002118:Calcium