Mapping Sites of Potential Proximity between the Dihydropyridine Receptor and RyR1 in Muscle Using a Cyan Fluorescent Protein-Yellow Fluorescent Protein Tandem as a Fluorescence Resonance Energy Transfer Probe

Abstract

Excitation-contraction coupling in skeletal muscle involves conformational coupling between the dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) at junctions between the plasma membrane and sarcoplasmic reticulum. In an attempt to find which regions of these proteins are in close proximity to one another, we have constructed a tandem of cyan and yellow fluorescent proteins (CFP and YFP, respectively) linked by a 23-residue spacer, and measured the fluorescence resonance energy transfer (FRET) of the tandem either in free solution or after attachment to sites of the ␣ 1S and ␤ 1a subunits of the DHPR. For all of the sites examined, attachment of the CFP-YFP tandem did not impair function of the DHPR as a Ca 2؉ channel or voltage sensor for excitation-contraction coupling. The free tandem displayed a 27.5% FRET efficiency, which decreased significantly after attachment to the DHPR subunits. At several sites examined for both ␣ 1S (N-terminal, proximal II-III loop of a two fragment construct) and ␤ 1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (␣ 1S -null) or dyspedic (RyR1-null) myotubes. However, compared with dysgenic myotubes, the FRET efficiency in dyspedic myotubes increased from 9.9 to 16.7% for CFP-YFP attached to the N-terminal of ␤ 1a , and from 9.5 to 16.8% for CFP-YFP at the C-terminal of ␣ 1S . Thus, the tandem reporter suggests that the C terminus of ␣ 1S and the N terminus of ␤ 1a may be in close proximity to the ryanodine receptor.

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

“…Previous work had shown that attachment of either a CFP-YFP tandem (25), or the biotin acceptor domain (26), to a C-terminally truncated ␣ 1S (at residue 1667) did not interfere with function after expression in dysgenic myotubes, and we found similar results for truncation and attachment of YPet at residue 1666 (data not shown). Additionally, we observed that constructs C-terminally tagged with YPet at residue 1666 and N-terminally tagged with CyPet (CyPet-␣ 1S 1666-YPet) produced co-localized cyan and yellow puncta after expression in dysgenic myotubes (Fig.…”

“…On the basis of these results, all the C-terminally tagged ␣ 1S constructs used for the FRET measurements in this study were truncated at residue 1636. All other ␣ 1S constructs did not have this truncation and their sequence ended at residue 1860 (25).…”

“…There is also evidence suggestive of binding interactions between other DHPR cytoplasmic domains and RyR1. These include the ␣ 1S III-IV loop, which is known to modulate EC coupling (22,23), and the C terminus, a segment of which binds to RyR1 in vitro (24), and which becomes partially occluded in the presence of RyR1 in vivo (25,26).…”

Stac3 has a direct role in skeletal muscle-type excitation–contraction coupling that is disrupted by a myopathy-causing mutation

“…The construction of the expression plasmids for YFPCa V 1.1, unlabeled β 1a , and unlabeled Stac3 was described previously (14,25,26). To create Stac3-BFP, the Stac3 fragment from Stac3-YFP (14) was inserted into pmTagBFP2-N1 (Addgene) using the restriction enzymes NdeI + BamHI.…”

El acoplamiento excitación-contracción en el músculo esquelético: preguntas por responder a pesar de 50 años de estudio

“…En el caso del músculo esquelético, se han podido identificar sparks en anfibios, en embriones de mamífero y en miotubos en cultivo (65). Además, el empleo de la microscopía confocal de barrido láser en combinación con novedosas técnicas como la transferencia de energía por resonancia de fluorescencia (FRET) y la relación del desplazamiento del espectro de emisión/excitación (SEER) han permitido, entre otros hallazgos, estudiar el acoplamiento entre los receptores de dihidropiridinas y los de rianodina, y lograr imágenes confocales que permiten cuantificar el Ca 2+ dentro del retículo sarcoplásmico, respectivamente (66,67).…”