Abstract
Auxiliary channel subunits regulate membrane expression and modulate current properties of voltage-activated Ca(2+) channels and thus are involved in numerous important cell functions, including muscle contraction. Whereas the importance of the alpha(1S), beta(1a), and gamma Ca(2+) channel subunits in skeletal muscle has been determined by using null-mutant mice, the role of the alpha(2)delta-1 subunit in skeletal muscle is still elusive. We addressed this question by small interfering RNA silencing of alpha(2)delta-1 in reconstituted dysgenic (alpha(1S)-null) myotubes and in BC3H1 skeletal muscle cells. Immunofluorescence labeling of the alpha(1S) and alpha(2)delta-1 subunits and whole cell patch clamp recordings demonstrated that triad targeting and functional expression of the skeletal muscle Ca(2+) channel were not compromised by the depletion of the alpha(2)delta-1 subunit. The amplitudes and voltage dependences of L-type Ca(2+) currents and of the depolarization-induced Ca(2+) transients were identical in control and in alpha(2)delta-1-depleted muscle cells. However, alpha(2)delta-1 depletion significantly accelerated the current kinetics, most likely by the conversion of slowly activating into fast activating Ca(2+) channels. Reverse transcription-PCR analysis indicated that alpha(2)delta-1 is the exclusive isoform expressed in differentiated BC3H1 cells and that depletion of alpha(2)delta-1 was not compensated by the up-regulation of any other alpha(2)delta isoform. Thus, in skeletal muscle the Ca(2+) channel alpha(2)delta-1 subunit functions as a major determinant of the characteristic slow L-type Ca(2+) current kinetics. However, this subunit is not essential for targeting of Ca(2+) channels or for their primary physiological role in activating skeletal muscle excitation-contraction coupling.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) TPA BK005394
We addressed this question by small interfering RNA silencing of ␣2␦-1 in reconstituted dysgenic (␣1S-null) myotubes and in BC3H1 skeletal muscle cells
Null-mutant mice exist for all skeletal muscle isoforms of the Ca2ϩ channel subunits with the exception of ␣2␦-1
Summary
Design of siRNA Plasmids—siRNA target sequences corresponding to the ␣2␦-1 coding region (Cacna2d1, GenBankTM accession number NM_009784) were selected as recommended [13], and siRNAs targeting all known ␣2␦-1 splice variants [9] were expressed as hairpins from the pSilencer1.0-U6 siRNA expression vector (Ambion Ltd., Huntington, Cambridgeshire, UK). Quantitative analysis of ␣2␦-1 expression and clustering was performed by systematically screening of cover glasses for well differentiated transfected (GFP-positive) myotubes (GLT) or myoblasts (BC3H1) and classifying their clustered ␣2␦-1 expression (in the red channel) as strong (ϩϩϩ), medium (ϩϩ), weak (ϩ), or absent (Ϫ), whereby strong labeling was defined as the staining intensity and density of clusters observed in the majority of the control myotubes. The nucleotide sequence data of the putative mouse ␣2␦-4 subunit were submitted to GenBankTM and are available in the Third Party Annotation Section of the DDBJ/ EMBL/GenBankTM data bases under the accession number TPA BK005394
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