FRET-Based Structural Analysis of the Skeletal Muscle DHPR using Biarsenical Labeling

The α1s skeletal muscle dihydropyridine receptor (DHPR) subunit triggers muscle contraction during excitation contraction (EC) coupling. The intracellular loops of this voltage-gated calcium channel have diverse functions, though the spatial organization of these loops relative to each other and to other DHPR subunits, is poorly understood. We employed a FRET-based assay to determine the proximity of these intracellular loops relative to the N-terminus of the α1s DHPR subunit and then determined how these proximities changed upon binding of the β1a DHPR subunit. Recombinant α1s DHPRs were expressed in HEK293T cells and then FRET was measured from YFP fused to the DHPR N-terminus to the biarsenical reagent ReAsH targeted to a tetracysteine (Tc) tag inserted into each of the DHPR intracellular loops. Energy transfer was highest to ReAsH bound to a Tc tag placed adjacent to the N-terminally fused YFP (E∼40-50%) whereas low FRET (E∼10%) was observed for a negative control construct lacking a Tc tag. Placement of Tc tags in each of the DHPR intracellular loops resulted in intermediate FRET efficiencies whereas insignificant energy transfer was observed between the DHPR N- and C-termini. Addition of purified recombinant DHPR β1a subunit resulted in significant enhancement of energy transfer between the N-terminal YFP and ReAsH targeted to the DHPR α1s II-III loop, a major EC coupling determinant. Our data demonstrate that FRET measurements using biarsenical labeling reagents offer an effective approach to study DHPR structure since disruption of native conformation is minimal. In addition, these studies should allow for subsequent measurements of changes in DHPR structure that occur during EC coupling.Supported by NIH grants R01AR059126 (to JDF) and R03AR066359 (to CFP).