title: Bimolecular fluorescence complementation and targeted biotinylation provide insight into the topology of the skeletal muscle Ca ( 2+) channel β1a 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 β1a subunits associated with native CaV 1.1 in DHPRs of myotubes. The construct YN-β1a-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 β1a, was fully functional and displayed yellow fluorescence within DHPR tetrads after expression in β1-knockout (β1KO) myotubes; this yellow fluorescence demonstrated the occurrence of BiFC of YN and YC on the β1a 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-β1a-YC in DHPR tetrads appeared to be intramolecular between N- and C-termini of individual β1a subunits rather than between adjacent DHPRs because BiFC (1) was observed for YN-β1a-YC co-expressed with CaV 1.2 (which does not form tetrads) and (2) was not observed after co-expression of YN-β1a-YN plus YC-β1a-YC in β1KO myotubes. Thus, β1a 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 β1a subunits in DHPR tetrads.
authors.affiliation: Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Sheridan
authors.forename: Ong
authors.initials: O
authors.lastname: Moua
authors.forename: Nancy M
authors.initials: NM
authors.lastname: Lorenzon
authors.forename: Kurt G
authors.initials: KG
authors.lastname: Beam
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001710:Biotin / Q000235:genetics; D019921:Biotinylation; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002460:Cell Line; D005453:Fluorescence; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology*; D021122:Protein Subunits / Q000502:physiology; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D020746:Calcium Channels, L-Type; D008164:Luminescent Proteins; D021122:Protein Subunits; C054562:yellow fluorescent protein, Bacteria; D001710:Biotin