Does the voltage-gated calcium channel α₂δ-1 subunit play a dual function in skeletal muscle?

The dihydropyridine receptor (DHPR), a high voltage-activated calcium (Ca²⁺) channel, represents a key element in the excitation–contraction (EC) coupling machinery of muscle cells. In skeletal muscle, the DHPR fulfils two known functions: (i) it controls the ryanodine receptor activity, which makes it commonly referred to as the voltage-sensor for EC coupling and (ii) it supports an L-type voltage-activated Ca²⁺ current. The DHPR is a macromolecular complex composed of the Ca_v1.1 pore-forming subunit, surrounded by β_1a, γ₁ and α₂δ-1 auxiliary subunits. Whereas knock-out animals have clearly demonstrated the essential role of Ca_v1.1 and β_1a subunits in EC coupling and in the control of the Ca²⁺ conductance, the functional importance of α₂δ-1 in skeletal muscle remains largely elusive, despite the lethal phenotype of α₂δ-1-null embryos. Interestingly, down-regulation of the α₂δ-1 protein in the BC3H1 muscle cell line with siRNA only resulted in acceleration of Ca²⁺ current activation kinetics with no effect on both EC coupling and Ca_v1.1 trafficking (Obermair et al. 2005). However, it is hard to understand how alteration of Ca²⁺ current kinetics could be the cause of the lethal phenotype observed in α₂δ-1-null mice embryos; instead α₂δ-1 is most likely to be involved in other cell functions independent of the DHPR activity. Previous observations suggested such a function: (i) α₂δ-1 appears early during muscle development, at a period when the Ca_v1.1 subunit is not yet expressed, and (ii) sequence analysis of α₂δ-1 revealed the presence of consensus domains, known to be involved in cell adhesion and protein–protein interactions (see Fig. 1 for structural organization of the α₂δ-1 subunit). In a recent issue of The Journal of Physiology, García et al. raised the possibility that α₂δ-1 could mediate the interaction of muscle cells with the extracellular matrix (Garcia et al. 2008). Using immunohistochemistry and a siRNA knock-down strategy, the authors provided new insights into the functional involvement of α₂δ-1 in skeletal muscle cells.

Figure 1. Structural organization of the voltage-gated Ca²⁺ channel α₂δ-1 subunit and its potential functions in the skeletal muscle.

In skeletal muscle, the dihydropyridine receptor (DHPR) is composed of the Ca_v1.1 subunit (blue), surrounded by auxiliary β1_a (red), γ (orange) and α₂δ-1 (green) subunits. The α₂δ-1 subunit is the product of a unique gene, post-translationally cleaved into α2 and δ peptides remaining associated by disulphide bonds (S–S), and highly glycosylated. On the basis of hydrophobicity studies and direct-site mutagenesis, it was proposed that the α2 subunit is completely extracellular, while a unique transmembrane helix formed by the carboxy-terminale region of the δ subunit allows the anchoring of α₂δ-1 at the plasma membrane. Sequence analysis revealed the presence of a von Willebrand factor type A (VWA) domain, habitually involved in divalent-cation-dependent interactions with the extracellular matrix. Two Cache domains have also been identified and could be relevant for gabapentinoid drugs binding to the α₂δ-1 subunit.

In order to precisely examine the expression pattern of α₂δ-1 and Ca_v1.1 subunits during skeletal muscle development, the authors developed an affinity-purified rabbit polyclonal antibody against α₂δ-1. When used in combination with the Ca_v1.1 mouse monoclonal antibody, this anti-α₂δ-1 allows simultaneous separate detection of Ca_v1.1 and α₂δ-1 proteins in the same cell, which was not achieved in previous studies (Obermair et al. 2005). Western blot analysis performed on whole-cell homogenates of either primary culture myotubes or COS7 cells transfected with the cDNA encoding for α₂δ-1 revealed that this antibody specifically recognizes a 140 kDa protein which is not detected in non-transfected COS7 cells (Garcia et al. 2008, their Fig. 1C), providing evidence for the specificity of the α₂δ-1 antibody. Double staining of adult skeletal muscle tissue revealed a co-localization of α₂δ-1 with Ca_v1.1 (García et al., their Fig. 1B), consistent with previous reports showing localization of α₂δ-1 in skeletal muscle triads (Obermair et al. 2005). However, this expression pattern is clearly dependent on the developmental stage of skeletal muscle cells. Indeed, in 2-day-old myotubes, while Ca_v1.1 localizes close to the centre of the cells, α₂δ-1 predominantly appears at the ends of the myotubes from where Ca_v1.1 is largely excluded. During in vitro development, α₂δ-1 then gradually becomes more homogeneously distributed throughout the cells (6-day- and 8-day-old myotubes), to finally present an expression pattern similar to that of Ca_v1.1 in 12-day-old myotubes (García et al., their Fig. 2). The exclusive localization of α₂δ-1 at the ends of the myotubes during early developmental stages strongly suggests that this protein is involved in a mechanism other than EC coupling or Ca²⁺ current regulation. To further investigate the functional role of α₂δ-1 during cell maturation, siRNAs were used in the C2C12 myogenic cell line to down-regulate the expression level of the protein. Based on the efficiency of the down-regulation level of the α₂δ-1 protein, a unique siRNA sequence (cloned into the pSUPER.neo vector which confers neomycin resistance to transfected cells) was chosen among five different ones, and transfected into C2C12 cells (García et al., their Fig. 3). Transfected cells were then selected by adding the G418 neomycin analogue into the culture medium 24 h after transfection; the capability of cells to migrate in a region of interest was then evaluated 18 h later. Under these experimental conditions, α₂δ-1 siRNA-transfected cells (pS-α₂δ-1) presented a drastic impairment of migration capacity as compared to cells transfected with a scrambled siRNA (pS-Ctr) (García et al., their Fig. 4). In order to further investigate the possible role of α₂δ-1 in extracellular signalling, the capacity of α₂δ-1 knock-down cells to attach and spread on the substrate was also studied. Again, pS-α₂δ-1-transfected C2C12 myoblasts presented a clear defect of attachment as compared to pS-Ctr cells during either a 30 or 60 min adhesion trial (García et al., their Fig. 5A and B). A viability test on non-adherent cells revealed that this alteration in myoblast attachment was due to the absence of α₂δ-1 and not to a higher mortality of pS-α₂δ-1-transfected myoblasts as compared to control cells. Spreading capacity of pS-α₂δ-1-transfected myoblasts was also studied and was found to be reduced by about half in comparison with that of control cells (García et al., their Fig. 5C). Taken together, these results convincingly suggest that α₂δ-1 is involved in cell adhesion and spreading. Finally, in order to determine whether defects in myoblast attachment, migration and spreading are directly related to the absence of α₂δ-1 or rather to an alteration of the L-type Ca²⁺ current, patch clamp recordings were performed in the whole-cell configuration. Voltage-activated Ca²⁺ currents with similar properties were recorded in pS-α₂δ-1- and in pS-Ctr-transfected cells, suggesting that the absence of the α₂δ-1 protein in C2C12 cells at a myoblastic stage does not affect the ionic function of the DHPR (García et al., their Fig. 6). This result confirms that the phenotype observed in α₂δ-1-deficient C2C12 myoblasts is well mediated by the direct absence of the protein.

In conclusion, the authors demonstrate a novel function of the voltage-gated Ca²⁺ channel α₂δ-1 subunit in skeletal muscle cells, independent of the DHPR regulation. They show that down-regulation of the α₂δ-1 protein in C2C12 myoblasts results in a drastic alteration of cell attachment, migration and spreading, independent of any modification of the L-type Ca²⁺ current, suggesting that α₂δ-1 can mediate a direct interaction of the cell with the extracellular matrix. This result is of primary importance since it is the first demonstration of a functional role of α₂δ-1 unrelated to the DHPR function. Besides involvement in cellular binding with collagen-coated substrate, which was clearly demonstrated in this work, α₂δ-1 is likely to be involved in vivo in more complex interactions with other molecules by means of its extracellular domains. However, some questions remain to be elucidated. First, it would be interesting to better characterize the interaction of α₂δ-1 with the extracellular matrix, and to identify the molecular determinants involved in this interaction. Simultaneous down-regulation of the wild-type α₂δ-1 protein and over-expression of a mutated and/or truncated form in C2C12 cells could allow this characterization. Furthermore, although α₂δ-1 does not seem to be implicated in the L-type Ca²⁺ current regulation of C2C12 myoblasts, a physiological contribution of the protein in the control of the channel function of the DHPR at a more mature stage cannot be ruled out. It was recently proposed that the slow voltage-activated Ca²⁺ current could play a role in the fusion of satellite cells (Luin & Ruzzier, 2007) as well as in the aggregation of acetylcholine receptors during postsynaptic development of skeletal muscle cells (Milholland et al. 2007). Since down-regulation of the α₂δ-1 subunit has already been reported to alter Ca²⁺ current kinetics (Obermair et al. 2005), characterization of the properties of these two processes in the absence of the protein could bring critical information with regards to the physiological participation of the voltage-activated Ca²⁺ current in skeletal muscle. Finally, the authors stipulate that α₂δ-1, by promoting cell attachment, migration and spreading, may be crucial for muscle development, muscle repair and other processes at times when myoblasts attachment and migration are fundamental. However, the use of C2C12 myoblasts weakly takes into account these developmental processes. Down-regulation of the α₂δ-1 protein in primary culture myoblasts before induction of fusion and differentiation into myotubes represents a significant step towards better understanding the functional importance of α₂δ-1 in muscle development. A closely related functional characterization was recently performed by the group of P. D. Allen (Gach et al. 2008). Surprisingly, and in contrast to what one may have expected, no alteration in myoblast division and in myotube growth and differentiation was observed in α₂δ-1 knock-down cells, despite the almost complete absence of the protein. Moreover, that study also revealed that, although the α₂δ-1 subunit is not essential for EC coupling (see also Obermair et al. 2005), it nevertheless plays an important role in maintaining Ca²⁺ transients in response to prolonged depolarizations or repeated trains of action potentials, i.e. well within the physiolological operating mode of functioning muscle cells. Along this idea, the observation that α₂δ-1 binding with gabapentinoid drugs affects Ca²⁺ currents in myotubes (Alden & Garcia, 2002) may also be speculated to underly related pharmacological effects on skeletal muscle function.

Hence, if the involvement of the α₂δ-1 subunit in the conductive function of the DHPR as well as indirectly in EC coupling seems to be relatively clear, its role in extracellular signalling needs to be further investigated before α₂δ-1 can be established as a key participant in signalling for muscle development.