title: Distinct Components of Retrograde Ca(V)1.1-RyR1 Coupling Revealed by a Lethal Mutation in RyR1.
abstract: The molecular basis for excitation-contraction coupling in skeletal muscle is generally thought to involve conformational coupling between the L-type voltage-gated Ca(2+) channel (CaV1.1) and the type 1 ryanodine receptor (RyR1). This coupling is bidirectional; in addition to the orthograde signal from CaV1.1 to RyR1 that triggers Ca(2+) release from the sarcoplasmic reticulum, retrograde signaling from RyR1 to CaV1.1 results in increased amplitude and slowed activation kinetics of macroscopic L-type Ca(2+) current. Orthograde coupling was previously shown to be ablated by a glycine for glutamate substitution at RyR1 position 4242. In this study, we investigated whether the RyR1-E4242G mutation affects retrograde coupling. L-type current in myotubes homozygous for RyR1-E4242G was substantially reduced in amplitude (∼80%) relative to that observed in myotubes from normal control (wild-type and/or heterozygous) myotubes. Analysis of intramembrane gating charge movements and ionic tail current amplitudes indicated that the reduction in current amplitude during step depolarizations was a consequence of both decreased CaV1.1 membrane expression (∼50%) and reduced channel Po (∼55%). In contrast, activation kinetics of the L-type current in RyR1-E4242G myotubes resembled those of normal myotubes, unlike dyspedic (RyR1 null) myotubes in which the L-type currents have markedly accelerated activation kinetics. Exogenous expression of wild-type RyR1 partially restored L-type current density. From these observations, we conclude that mutating residue E4242 affects RyR1 structures critical for retrograde communication with CaV1.1. Moreover, we propose that retrograde coupling has two distinct and separable components that are dependent on different structural elements of RyR1.
authors.affiliation: Cardiology Division, Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado. Electronic address: roger.bannnister@ucdenver.edu.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Bannister
authors.affiliation: Department of Biology and Earth Science, Otterbein University, Westerville, Ohio.
authors.forename: David C
authors.initials: DC
authors.lastname: Sheridan
authors.affiliation: Department of Physiology and Biophysics, University of Colorado School of Medicine, Aurora, Colorado. Electronic address: kurt.beam@ucdenver.edu.
authors.forename: Kurt G
authors.initials: KG
authors.lastname: Beam
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D055724:Electrophysiological Phenomena / Q000187:drug effects; D005786:Gene Expression Regulation; D007700:Kinetics; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: C529218:CACNA1S protein, mouse; D020746:Calcium Channels, L-Type; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C000592747:ryanodine receptor 1, mouse