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

Symeon Papadopoulos,Valérie Leuranguer,Roger A Bannister,Kurt G Beam

doi:10.1074/jbc.m405317200

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 alpha1S and beta1a 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 Ca2+ 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 alpha1S (N-terminal, proximal II-III loop of a two fragment construct) and beta1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (alpha1S-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 beta1a, and from 9.5 to 16.8% for CFP-YFP at the C-terminal of alpha1S. Thus, the tandem reporter suggests that the C terminus of alpha1S and the N terminus of beta1a may be in close proximity to the ryanodine receptor.

Highlights

A variety of evidence indicates that excitation-contraction (EC)1 coupling in skeletal muscle involves conformational coupling between dihydropyridine receptors in the plasma membrane and type 1 ryanodine receptors (RyR1) in the sarcoplas
By measuring the fluorescence resonance energy transfer (FRET) efficiency of a CFP-YFP tandem either free or fused to sites of the dihydropyridine receptor (DHPR), we have examined how the environment of these sites changes when the DHPR is inserted into plasma membrane at junctions with the SR
An additional decrease in FRET efficiency occurred when either CFP-YFP-␤1a or ␤1aCFP-YFP was inserted into junctions

Summary

Introduction

A variety of evidence indicates that excitation-contraction (EC) coupling in skeletal muscle involves conformational coupling between dihydropyridine receptors in the plasma membrane and type 1 ryanodine receptors (RyR1) in the sarcoplas-. Other biochemical studies showed an interaction between RyR1 residues 3609 and 3643 and the proximal portion (1393–1527) of the ␣1S C-terminal [19] If this interaction occurs in vivo, it cannot be a sole determinant of skeletal-type EC coupling because replacing the II-III loop of ␣1C with that of ␣1S is sufficient to convert EC coupling from cardiac-type (Ca2ϩ-entry dependent) to skeletal-type, independent of the identity (cardiac versus skeletal) of the C-terminal [20]. A subset of ␣1S residues (720 –765) in the II-III loop have been shown to be both critical for bidirectional signaling [21] and to interact very weakly with RyR1 residues 1837–2168 in yeast two-hybrid studies [22] This interaction seems unlikely to be the sole determinant of skeletal type coupling because (i). In the accompanying article [29], we describe another approach for probing the topology of junctions

Objectives

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Conclusion