The β-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 β-subunit in the expression of L-type Ca2+ current and charge movements in RyR1 knock-out (KO), β1 KO, and double, β1/RyR1 KO myotubes generated by mating heterozygous, β1 KO and RyR1 KO mice. Primary myotube cultures of each genotype were transfected with various, β-isoforms and then whole-cell voltage-clamped for measurements of Ca2+ and gating currents. Overexpression of the endogenous skeletal, β1a isoform resulted in a low-density Ca2+ current either in RyR1 KO (36 ± 9 pS/pF) or in β1/RyR1 KO (34 ± 7 pS/pF) myotubes. However, the heterologous, β2a 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, β1/RyR1 KO (115 ± 18 pS/pF) myotubes. Other variants tested from the four, β gene families (β1a, β1b, β1c, β3, and β4) were unable to enhance Ca2+ current expression in RyR1 KO myotubes. In contrast, intramembrane charge movements in β2a-expressing β1a/RyR1 KO myotubes were significantly lower than in β1a-expressing β1a/RyR1 KO myotubes, and the same tendency was observed in the RyR1 KO myotube. Thus, β2a had a preferential ability to recover Ca2+ current, whereas β1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (β2a C3,4S) eliminated the RyR1-independent Ca2+ current expression. Furthermore, Ca2+ current enhancement was observed with a β2a variant lacking the double cysteine motif and fused to the surface membrane glycoprotein CD8. Thus, tethering the β2a 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 β1a-subunit is critically involved in process.
|Original language||English (US)|
|Number of pages||18|
|Issue number||2 I|
|State||Published - Feb 1 2003|
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