The calcium channel α2/δ1 subunit is involved in extracellular signalling

Abstract

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.

The calcium channel α2/δ1 subunit interacts with ATP5b in the plasma membrane of developing muscle cells

“…The discrepancy in subunit levels has been demonstrated both at the mRNA (38) and the protein levels (23), suggesting that ␣2/␦1 may have a role in skeletal muscle independent from the DHPR complex. In agreement with those results, we have found that the ␣2/␦1 subunit localizes at the ends of cells with little or no association with ␣ 1 1.1 subunits in myotubes after 2 days of induction of fusion and differentiation (15). With longer times in differentiation medium, the localization of ␣2/␦1 gradually becomes homogeneous until it colocalizes almost completely with ␣ 1 1.1.…”

“…The association of ␣2/␦1 and ATP5b occurs at both the plasma membrane and in intracellular membranes, as demonstrated by protein labeling with biotin and antibodies, and FRET analysis. We have previously shown that the ␣2/␦1 subunit does not always colocalize with the ␣ 1 1.1 subunit and that it is involved in attachment and migration of myoblasts (15). Thus we hypothesized that the ␣2/␦1 subunit may be interacting with other cellular components different from DHPRs early during development.…”

“…Reducing ␣2/␦1 levels with small interfering RNA in muscle cells resulted in an acceleration of the calcium current with dissimilar effects on current conductance. Reduction of ␣2/␦1 in nonfusing BC3H1 or in C2C12 muscle cells had a small effect on current conductance (15,25), whereas in transduced myotubes the conductance was significantly reduced (13). In myotubes from the dysgenic cell line GLT, reduction of ␣2/␦1 levels did not modify the amplitude or the voltage dependence of calcium transients examined under voltage clamp (25).…”

Evidence for MOR on cell membrane, sarcoplasmatic reticulum and mitochondria in left ventricular myocardium in rats

“…Double immunofluorescence staining was processed as described previously [25,26]. The sections mounted in gelatin-coated slides were incubated overnight with the following primary antibodies: rabbit polyclonal anti-MOR (1:1000) (gift from S. Schulz and V. Höllt, Magdeburg, Germany) [4] alone or in combination with (a) mouse monoclonal antidihydropyridine receptor (ɑ2 subunit) (1:1000) to identify the voltage-gated L-type Ca 2+ channel (anti-Ca v 1.2) (SIGMA ® , Missouri, USA) [27][28][29], (b) mouse monoclonal anti-Inositol-1,4,5-trisphosphate receptor type III (1:600) to identify ryanodine receptors (RyR) of the sarcoplasmatic reticulum (BD Biosciences ® , Europe) [30,31], or (c) rat anti-mitochondrium marker MTC02 (1:300) to identify mitochondrial structures (Thermo Scientific) [32]. The rabbit polyclonal anti-MOR is known to be directed against the amino acids 386-398 of the MOR [4] and also known to recognize an expected protein band at 55 kDa in Western blot analysis of MOR transfected HEK293 cells [33] and mouse brain tissue [34].…”

Temporal Expression of Calcium Channel Subunits in Satellite Cells and Bone Marrow Mesenchymal Cells

“…Interestingly, the subunits of skeletal muscle L-type calcium channel are not expressed simultaneously during differentiation of myotubes in vitro. We have shown that the α2δ1 subunit is the first one to be expressed in freshly dissociated satellite cells and is followed by expression of the α1, β, and γ subunits [6,7]. Further, we have shown that more than 50 % of freshly isolated satellite cells express α2δ1 subunit and that these cells commit to the muscle lineage to form myotubes [7].…”