title: Ca2+ current and charge movements in skeletal myotubes promoted by the beta-subunit of the dihydropyridine receptor in the absence of ryanodine receptor type 1.
abstract: The beta-subunit of the dihydropyridine receptor (DHPR) enhances the Ca(2+) channel and voltage-sensing functions of the DHPR. In skeletal myotubes, there is additional modulation of DHPR functions imposed by the presence of ryanodine receptor type-1 (RyR1). Here, we examined the participation of the beta-subunit in the expression of L-type Ca(2+) current and charge movements in RyR1 knock-out (KO), beta1 KO, and double beta1/RyR1 KO myotubes generated by mating heterozygous beta1 KO and RyR1 KO mice. Primary myotube cultures of each genotype were transfected with various beta-isoforms and then whole-cell voltage-clamped for measurements of Ca(2+) and gating currents. Overexpression of the endogenous skeletal beta1a isoform resulted in a low-density Ca(2+) current either in RyR1 KO (36 +/- 9 pS/pF) or in beta1/RyR1 KO (34 +/- 7 pS/pF) myotubes. However, the heterologous beta2a variant with a double cysteine motif in the N-terminus (C3, C4), recovered a Ca(2+) current that was entirely wild-type in density in RyR1 KO (195 +/- 16 pS/pF) and was significantly enhanced in double beta1/RyR1 KO (115 +/- 18 pS/pF) myotubes. Other variants tested from the four beta gene families (beta1a, beta1b, beta1c, beta3, and beta4) were unable to enhance Ca(2+) current expression in RyR1 KO myotubes. In contrast, intramembrane charge movements in beta2a-expressing beta1a/RyR1 KO myotubes were significantly lower than in beta1a-expressing beta1a/RyR1 KO myotubes, and the same tendency was observed in the RyR1 KO myotube. Thus, beta2a had a preferential ability to recover Ca(2+) current, whereas beta1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (beta2a C3,4S) eliminated the RyR1-independent Ca(2+) current expression. Furthermore, Ca(2+) current enhancement was observed with a beta2a variant lacking the double cysteine motif and fused to the surface membrane glycoprotein CD8. Thus, tethering the beta2a variant to the myotube surface activated the DHPR Ca(2+) current and bypassed the requirement for RyR1. The data suggest that the Ca(2+) current expressed by the native skeletal DHPR complex has an inherently low density due to inhibitory interactions within the DHPR and that the beta1a-subunit is critically involved in process.
authors.affiliation: Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA.
authors.forename: Chris A
authors.initials: CA
authors.lastname: Ahern
authors.forename: David C
authors.initials: DC
authors.lastname: Sheridan
authors.forename: Weijun
authors.initials: W
authors.lastname: Cheng
authors.forename: Lindsay
authors.initials: L
authors.lastname: Mortenson
authors.forename: Priya
authors.initials: P
authors.lastname: Nataraj
authors.forename: Paul
authors.initials: P
authors.lastname: Allen
authors.forename: Michel
authors.initials: M
authors.lastname: De Waard
authors.forename: Roberto
authors.initials: R
authors.lastname: Coronado
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000502:physiology*; D002478:Cells, Cultured; D004553:Electric Conductivity; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel / Q000172:deficiency* / Q000502:physiology; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D021122:Protein Subunits; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium