Bimolecular fluorescence complementation and targeted biotinylation provide insight into the topology of the skeletal muscle Ca ( 2+) channel ?1a subunit.

ABSTRACT: 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 ?1a subunits associated with native CaV 1.1 in DHPRs of myotubes. The construct YN-?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 ?1a, was fully functional and displayed yellow fluorescence within DHPR tetrads after expression in ?1-knockout (?1KO) myotubes; this yellow fluorescence demonstrated the occurrence of BiFC of YN and YC on the ?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-?1a-YC in DHPR tetrads appeared to be intramolecular between N- and C-termini of individual ?1a subunits rather than between adjacent DHPRs because BiFC (1) was observed for YN-?1a-YC co-expressed with CaV 1.2 (which does not form tetrads) and (2) was not observed after co-expression of YN-?1a-YN plus YC-?1a-YC in ?1KO myotubes. Thus, ?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 ?1a subunits in DHPR tetrads.

SUBMITTER: Sheridan DC 

PROVIDER: S-EPMC3367678 | biostudies-literature | 2012 Jan-Feb

REPOSITORIES: biostudies-literature