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

The skeletal muscle dihydropyridine receptor (DHPR) in the t-tubular membrane serves as the Ca2+ channel and voltage sensor for excitation-contraction (EC) coupling, triggering Ca2+ release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR). The two proteins appear to be physically linked, and both the alpha(1S) and beta(1a) subunits of the DHPR are essential for EC coupling. Within alpha(1S), cytoplasmic domains of importance include the I-II loop (to which beta(1a) binds), the II-III and III-IV loops, and the C terminus. However, the spatial relationship of these domains to one another has not been established. Here, we have taken the approach of measuring FRET between fluorescent proteins inserted into pairs of alpha(1S) cytoplasmic domains. Expression of these constructs in dyspedic (RyR1 null) and dysgenic (alpha(1S) null) myotubes was used to test for function and targeting to plasma membrane/SR junctions and to test whether the presence of RyR1 caused altered FRET. We found that in the absence of RyR1, measureable FRET occurred between the N terminus and C terminus (residue 1636), and between the II-III loop (residue 626) and both the N and C termini; the I-II loop (residue 406) showed weak FRET with the II-III loop but not with the N terminus. Association with RyR1 caused II-III loop FRET to decrease with the C terminus and increase with the N terminus and caused I-II loop FRET to increase with both the II-III loop and N terminus. Overall, RyR1 appears to cause a substantial reorientation of the cytoplasmic alpha(1S) domains consistent with their becoming more closely packed.