Abstract
To study the interactions of the alpha1S subunit of the skeletal muscle L-type Ca2+ channel with the skeletal beta1a and the cardiac beta2a, these subunits were expressed alone or in combination in tsA201 cells. Immunofluorescence- and green fluorescent protein-labeling showed that, when expressed alone, beta1a was diffusely distributed throughout the cytoplasm, beta2a was localized in the plasma membrane, and alpha1S was concentrated in a tubular/reticular membrane system, presumably the endoplasmic reticulum (ER). Upon coexpression with alpha1S, beta1a became colocalized with alpha1S in the ER. Upon coexpression with beta2a, alpha1S redistributed to the plasma membrane, where it aggregated in large clusters. Coexpression of alpha1S with beta1a but not with beta2a increased the frequency at which cells expressed L-type currents. A point mutation (alpha1S-Y366S) or deletion (alpha1S-Delta351-380) in the beta interaction domain of alpha1S blocked both translocation of beta1a to the ER and beta2a-induced translocation of the alpha1S mutants to the plasma membrane. However, the point mutation did not interfere with beta1a-induced current stimulation. Thus, beta1a and beta2a are differentially distributed in tsA201 cells and upon coexpression with alpha1S, form alpha1S. beta complexes in different cellular compartments. Complex formation but not current stimulation requires the intact beta interaction domain in the I-II cytoplasmic loop of alpha1S.
Highlights
Eral membrane protein, the 1a subunit, and the ␥ subunit, which is unique for this tissue (6 – 8)
1a and ␣1S Are Expressed in Different Cellular Compartments—An expression plasmid encoding a fusion protein of the 1a subunit of the skeletal muscle DHP receptor and GFP, called GFP, has been constructed as a tool for studying the subcellular distribution of 1a transiently expressed in tsA201 cells. GFP aids in the identification of cells expressing the 1a subunit and allows its direct localization
A fusion protein of 1a and the green fluorescent protein and ␣1S constructs with modifications in the known  interaction domain in the cytoplasmic loop between repeats I and II of ␣1S have been generated
Summary
The skeletal muscle L-type Ca2ϩ channel is distinct from other voltage-gated Ca2ϩ channels with respect to its organization in the triad junction [29], with respect to its function in excitation-contraction coupling [4], and with respect to modulation of its channel properties [14, 30]. Whether interactions between the skeletal muscle ␣1 and  subunits expressed in mammalian non-muscle cells differ from those of other isoforms is not known. 1a and 2a on the subcellular distribution and current stimulation upon coexpression with ␣1S in tsA201 cells. A mutation in the  interaction domain of ␣1S interferes with the stable association of the subunits but not with the 1a-induced stimulation of Ca2ϩ currents
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