Bimolecular fluorescence complementation and targeted biotinylation provide insight into the topology of the skeletal muscle Ca2+ channel β1a subunit

In skeletal muscle, L-type calcium channels (DHPRs), localized to plasma membrane sarcoplasmic reticulum junctions, are tightly packed into groups of four termed tetrads. Here, we have used bimolecular fluorescence complementation (BiFC) and targeted biotinylation to probe the structure and organization of beta 1a subunits associated with native Ca(V)1.1 in DHPRs of myotubes. The construct YN-beta 1a-YC, in which the non-fluorescent fragments of YFP ("YN" corresponding to YFP residues 1-158, and "YC" corresponding to YFP residues 159-238) were fused, respectively, to the N- and C-termini of beta 1a, was fully functional and displayed yellow fluorescence within DHPR tetrads after expression in beta 1-knockout (beta 1KO) myotubes; this yellow fluorescence demonstrated the occurrence of BiFC of YN and YC on the beta 1a N- and C-termini. In these experiments, we avoided overexpression because control experiments in non-muscle cells indicated that this could result in non-specific BiFC. BiFC of YN-beta 1a-YC in DHPR tetrads appeared to be intramolecular between N- and C-termini of individual beta 1a subunits rather than between adjacent DHPRs because BiFC (1) was observed for YN-beta 1a-YC co-expressed with Ca(V)1.2 (which does not form tetrads) and (2) was not observed after co-expression of YN-beta 1a-YN plus YC-beta 1a-YC in beta 1KO myotubes. Thus, beta 1a function is compatible with N- and C-termini being close enough together to allow BiFC. However, both termini appeared to have positional freedom and not to be closely opposed by other junctional proteins since both were accessible to gold-streptavidin conjugates. Based on these results, a model is proposed for the arrangement of beta 1a subunits in DHPR tetrads.