The α2/δ1 subunit forms part of the dihydropyridine receptor, an essential protein complex for excitation-contraction (EC) coupling in skeletal muscle. Because of the lack of a viable knock-out animal, little is known regarding the role of the α2/δ1 subunit in EC coupling or in other cell functions. Interestingly, the α2/δ1 appears before the α1 subunit in development and contains extracellular conserved domains known to be important in cell signalling and inter-protein interactions. These facts raise the possibility that the α2/δ1 subunit performs vital functions not associated with EC coupling. Here, we tested the hypothesis that the α2/δ1 subunit is important for interactions of muscle cells with their environment. Using confocal microscopy, we followed the immunolocalization of α2/δ1 and α1 subunits with age. We found that in 2-day-old myotubes, the α2/δ1 subunit concentrated towards the ends of the cells, while the α1 subunit clustered near the centre. As myotubes aged (6-12 days), the α2/δ1 became evenly distributed along the myotubes and co-localized with α1. When the expression of α2/δ1 was blocked with siRNA, migration, attachment and spreading of myoblasts were impaired while the L-type calcium current remained unaffected. The results suggest a previously unidentified role of the α2/δ1 subunit in skeletal muscle and support the involvement of this protein in extracellular signalling. This new role of the α2/δ1 subunit may be crucial for muscle development, muscle repair and at times in which myoblast attachment and migration are fundamental.

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Most adult mammalian skeletal muscles contain only one isoform of ryanodine receptor (RyR1), whereas neonatal muscles contain two isoforms (RyR1 and RyR3). Membrane depolarization fails to evoke calcium release in muscle cells lacking RyR1, demonstrating an essential role for this isoform in excitation-contraction coupling. In contrast, the role of RyR3 is unknown. We studied the participation of RyR3 in calcium release in wild type (containing both RyR1 and RyR3 isoforms) and RyR3-/- (containing only RyR1) myotubes in the presence or absence of imperatoxin A (IpTxa), a high-affinity agonist of ryanodine receptors. IpTxa significantly increased the amplitude and the rate of release only in wild-type myotubes. Calcium currents, recorded simultaneously with the transients, were not altered with IpTxa treatment. [(3)H]ryanodine binding to RyR1 or RyR3 was significantly increased in the presence of IpTxa. Additionally, IpTxa modified the gating and conductance level of single RyR1 or RyR3 channels when studied in lipid bilayers. Our data show that IpTxa can interact with both RyRs and that RyR3 is functional in myotubes and it can amplify the calcium release signal initiated by RyR1, perhaps through a calcium-induced mechanism. In addition, our data indicate that when RyR3-/- myotubes are voltage-clamped, the effect of IpTxa is not detected because RyR1s are under the control of the dihydropyridine receptor.

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We examined developmental changes in calcium channel alpha2/delta subunit mRNA in skeletal muscle and their possible influence on L-type calcium currents (ICa-L). Several isoforms of alpha2/delta-1 mRNA were found in myotubes and muscle fibers, and their relative levels changed with time in culture or age of the animal. Levels of alpha2/delta-1a were largest in older myotubes and was the only alpha2/delta-1 isoform present in adult muscle. Both myotubes and muscle fibers also expressed low levels of alpha2/delta-2 and alpha2/delta-3 mRNA at all ages. alpha2/delta-4 mRNA could not be detected in either myotubes or muscle fibers. Changes in amplitude and voltage-dependent inactivation of the ICa-L concurred with the shift in alpha2/delta-1 isoform message, suggesting that alternative splicing of this subunit might be important for modulation of ICa-L.