Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor 1S Subunit Reveals Conformational Differences Induced by Binding of the β1a Subunit

The skeletal muscle dihydropyridine receptor (1S) subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous (1S) structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in (1S) subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each (1S) intracellular loop, with the highest FRET efficiencies measured to the (1S) II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the (1S) N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the (1a) dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that (1a) binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of (1a), which are essential to support EC coupling. Direct FRET measurements between (1S) and (1a) confirmed that both wild type and truncated (1a) bind similarly to (1S). These results provide new insights into the role of muscle-specific proteins on the structural arrangement of (1S) intracellular loops and point to a new conformational effect of the (1a) subunit in supporting skeletal muscle excitation-contraction coupling.