FRET Reveals Substantial Reorientation of the Cytoplasmic Interface of the Skeletal Muscle DHPR in the Presence of RyR1

In skeletal muscle, the dihydropyridine receptor (DHPR) in the t-tubular membrane serves as Ca2+channel and as voltage sensor for excitation-contraction (EC) coupling, triggering Ca2+release via a physical/conformational coupling to the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. The particulars of the structural and functional links between these two proteins are widely unknown. The putative intracellular portions of the DHPR a1S subunit, the N-terminus, C-terminus, and the loops connecting the four homologous repeats (I-IV), play important roles in the communication with the RyR1. Examples are the β-subunit recruiting function of the I-II loop, the bi-directional signaling function of the II-III loop with the RyR1 during EC-coupling, the influence of the III-IV loop on RyR1 mediated Ca2+delivery, and the a1S C-terminus. These channel parts are believed to either directly or indirectly interact with the RyR1, and the close spatial proximity between the two channels at t-tubule/SR ‘junctions’ constitutes the structural prerequisite for this linkage. The present work provides for the first time a structural insight into the arrangement of the crucial molecular components of the DHPR-RyR1 interaction, by using measurements of fluorescence resonance energy transfer (FRET), conducted within the cellular environment of living myotubes. Upon expression, the degree of FRET was determined for different combinations of labeled cytoplasmic a1S domains, using a sensitized emission FRET variant. Confocal fluorescence microscopy was applied to check for correct expression and function of the constructs upon expression in dyspedic (RyR1 null) and dysgenic (a1S null) myotubes. The presence of RyR1 significantly altered the intramolecular energy transfer for almost every double tagged a1S construct. These measurements reveal that virtually the complete cytoplasmic a1S architecture is significantly rearranged by the presence of the RyR1.