The calcium channel alpha2/delta1 subunit is involved in extracellular signalling

Abstract

The alpha2/delta1 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 alpha2/delta1 subunit in EC coupling or in other cell functions. Interestingly, the alpha2/delta1 appears before the alpha1 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 alpha2/delta1 subunit performs vital functions not associated with EC coupling. Here, we tested the hypothesis that the alpha2/delta1 subunit is important for interactions of muscle cells with their environment. Using confocal microscopy, we followed the immunolocalization of alpha2/delta1 and alpha1 subunits with age. We found that in 2-day-old myotubes, the alpha2/delta1 subunit concentrated towards the ends of the cells, while the alpha1 subunit clustered near the centre. As myotubes aged (6-12 days), the alpha2/delta1 became evenly distributed along the myotubes and co-localized with alpha1. When the expression of alpha2/delta1 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 alpha2/delta1 subunit in skeletal muscle and support the involvement of this protein in extracellular signalling. This new role of the alpha2/delta1 subunit may be crucial for muscle development, muscle repair and at times in which myoblast attachment and migration are fundamental.

Figure 1. Identification of α2/δ1a isoform by a polyclonal antibody

A, COS7 cells were transfected with a plasmid containing the full sequence of the α2/δ1a subunit, kindly provided by Drs N. Klugbauer and F. Hofmann and fixed 48 h later. Cells were examined for expression of the α2/δ1a protein by labelling with the α2/δ1 affinity-purified anti-1a isoform primary antibody (1 : 500) and Alexa-Fluor 488 goat anti-rabbit secondary antibody (1 : 1000). Confocal microscopy revealed diffuse staining of transfected cells. Control, untransfected, cells did not show any labelling. B, the tibialis anterior muscle was isolated from adult mice, frozen in liquid nitrogen, and sectioned. Sections of tissue were simultaneously labelled with the α2/δ1 affinity-purified anti-1a isoform antibody (1 : 500) and the α1 monoclonal antibody mAb 1A (1 : 1000; Morton & Froehner, 1987). The secondary antibodies were Alexa-Fluor 488 goat anti-rabbit and Alexa-Fluor 555 goat anti-mouse, both at 1 : 1000 (Molecular Probes). Examination of the sections with confocal microsopy showed co-localization of α2/δ1a and α1 labelling in a striated pattern, as would be expected for adult skeletal muscle. Note that α2/δ1a is the only isoform expressed in adult skeletal muscle. C, Western blot of whole-cell homogenate probed with the α2/δ1a polyclonal antibody. Two-day skeletal myotubes (C), COS7 cells transfected with α2/δ1a plasmid (1), and naïve COS7 cells (2). The affinity-purified antibody specifically recognizes both expressed and native α2/δ1a subunits. There was no signal in naïve COS7 cells.

Figure 2. Immunolocalization of α2/δ1 and α1 subunits in primary myotubes

Myotubes were simultaneously labelled with an affinity-purified α2/δ1 antibody and the α1 monoclonal antibody 1A at different times after fusion and differentiation were promoted. A, representative 2-day-old myotubes from different cultures showed a strong α2/δ1 labelling at the ends of the cells where α1 labelling was weak or absent. Bars, 10 μm. B–D, representative 6-, 8- and 12-day-old myotubes, respectively, showing a progressively more homogeneous localization of α2/δ1 and α1 subunits along the myotubes than the 2-day-old myotubes. The 12-day-old myotubes belong to different cultures. There were patches of α2/δ1 staining without α1 labelling in some myotubes still at 12 days. Calibration bars: B, 10 μm; C, 20 μm; D, 20 μm top and 10 μm bottom.

Figure 3. Measurement of siRNA-induced silencing in C2C12 cells

A, Western blot analysis of α2/δ1 subunit from whole-cell homogenate using monoclonal antibody mAb 20A. Control samples (C) were obtained from pS-Ctr-transfected cells. Numbers below the lanes correspond to the targets in the α2/δ1 sequence; (1) 252–270, (2) 536–554, (3) 763–781, (4) 1167–1185, and (5) 1462–1480. All samples were obtained from cells that had been selected with G418 after transfection of siRNA vectors. B, quantification of α2/δ1 protein reduction by siRNA expressed as per cent of control (pS-Ctr). Data correspond to the average of five measurements.

Figure 4. Migration is impaired in α2/δ1-deficient C2C12 cells

Images recorded right after the central area of the coverslip was cleared of myoblasts (0 h) and 18 h later. The last column represents the difference of the fields between 18 and 0 h. Images in each row were taken from the same place in the dish. Images in each pair correspond to Rhod-2 fluorescence or transmitted light. A, myoblasts deficient in α2/δ1 subunit (pS-α2/δ1) showed impaired migration to the denuded area, compared to control cells (pS-Ctr). Images are 625 μm × 625 μm. B, a significantly lower number of α2/δ1-deficient cells migrated to the denuded area of the coverslip after 18 h compared to control cells (P < 0.05).

Figure 5. Attachment and spreading is reduced in α2/δ1-deficient C2C12 cells

Control and α2/δ1-deficient cells were plated at 15 × 10³ cells cm⁻² and allowed to attach for 30 or 60 min to collagen I-coated coverslips. Unattached cells were rinsed. Cells were fixed and labelled with TO-PRO3. Random fields were recorded to quantify number of cells attached and area of the cells. A, a significantly lower number of α2/δ1-deficient C2C12 cells attached to the coverslips at both 30 and 60 min. Images are 625 μm × 625 μm. B, quantification of cell attachment for control (filled bars) or α2/δ1-deficient cells (open bars) (P < 0.05). C, spreading and consequently the area of α2/δ1-deficient cells was considerably smaller than in control cells. Images are 110 μm × 110 μm. (The online version of this figure is in colour.)

Figure 6. L-type calcium current is not modified in α2/δ1-deficient cells

Calcium currents recorded from C2C12 cells under the same experimental conditions as in Figs 4 and 5. A, representative L-type calcium currents from a control cell (pS-Ctr) and an α2/δ1-deficient cell (pS-α2/δ1). B, current–voltage relationship for all control (█) and α2/δ1-deficient (▴) cells. The smooth curves correspond to the fit of all data in each group to the equation described in the text.

Figure 7. Schematic representation of the interaction of α2/δ1 with the substratum and differential localization during myotube differentiation

Young myotubes (2 day) showed strong α2/δ1 labelling at the ends of the cells where α1 labelling was weak or absent. The top left represents a magnification of the end of the cell to illustrate the interaction of α2/δ1 with collagen. Older myotubes (12 day) showed homogeneous distribution of α2/δ1 along the cells and co-localization with α1. α2/δ1 is represented by squares and α1 by circles.