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

Abstract

2+ , calciumIn 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 b1a subunits associated with native Ca V 1.1 in DHPRs of myotubes. The construct YN-b1a-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 b1a, was fully functional and displayed yellow fluorescence within DHPR tetrads after expression in b1-knockout (b1KO) myotubes; this yellow fluorescence demonstrated the occurrence of BiFC of YN and YC on the b1a 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-b1a-YC in DHPR tetrads appeared to be intramolecular between Nand C-termini of individual b1a subunits rather than between adjacent DHPRs because BiFC (1) was observed for YN-b1a-YC co-expressed with Ca V 1.2 (which does not form tetrads) and (2) was not observed after co-expression of YN-b1a-YN plus YC-b1a-YC in b1KO myotubes. Thus, b1a 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 b1a subunits in DHPR tetrads.

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

“…The yellow fluorescence arising from the YFP tag was arrayed in double rows having a center-tocenter spacing of ϳ2 m, consistent with a T-tubular localization. Furthermore, the red fluorescence arising from the bound fluoro-gold streptavidin colocalized with the yellow fluorescence, indicating that there is effective biotinylation of the BAD attached to the ␤ 1a C terminus and that its disposition in triad junctions makes it accessible to a moderately sized (1 nm) probe, consistent with previous work on myotubes (34,45,46). This previous work also showed that EC coupling was not impaired when streptavidin bound to YFP-␤-BAD in myotubes (45).…”

Fluorescence Resonance Energy Transfer (FRET) Indicates That Association with the Type I Ryanodine Receptor (RyR1) Causes Reorientation of Multiple Cytoplasmic Domains of the Dihydropyridine Receptor (DHPR) α1S Subunit

“…Relevant to this issue, earlier experiments examined the spatial interrelationships of ␤ 1a subunits in DHPR tetrad arrays by analyzing constructs expressed in ␣ 1 null myotubes. These experiments failed to detect either intermolecular FRET (39) or bimolecular fluorescence complementation (40) between ␤ 1a subunits in adjacent DHPRs. Thus, it was suggested that the adjacent ␤ 1a subunits were separated from one another by Ͼ10 nm (see Fig.…”

“…8 in Ref. 40). If this suggested arrangement is correct, then the I-II loops of ␣ 1S also would likely be positioned at a distance Ͼ10 nm from the ␣ 1S cytoplasmic domains of adjacent DHPRs.…”

Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

“…For clarity, the β 1a subunits are superimposed on the α 1S subunits, and the α 2 δ-1 subunits, γ 1 subunits, and other nonessential components of the junction have been omitted. The orientation of β 1a within the tetrad follows on previous work ( Leuranguer et al, 2006 ; Sheridan et al, 2012 ). In the right panels (B and C), we present two potential mechanisms by which Rem (black ovals) may disrupt EC coupling.…”