Ca2+ current and charge movements in skeletal myotubes promoted by the β-subunit of the dihydropyridine receptor in the absence of ryanodine receptor type 1

The beta-subunit of the dihydropyridine receptor (DHPR) enhances the Ca2+ 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 G-subunit in the expression of L-type Ca2+ 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 Ca2+ and gating currents. Overexpression of the endogenous skeletal beta1a isoform resulted in a low-density Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ current, whereas beta1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (beta2a C3,4S) eliminated the RyR1-independent Ca2+ current expression. Furthermore, Ca2+ 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 Ca2+ current and bypassed the requirement for RyR1. The data suggest that the Ca2+ 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.