In skeletal muscle, Ca V 1.1 in the plasma membrane is physically linked to RyR1 in the SR, causing Ca V 1.1 to be arrayed in groups of four (tetrads) with the result that depolarization-induced conformational changes of Ca V 1.1 activate calcium release via RyR1. The identity of the underlying sites of protein-protein interaction, and whether they are direct or indirect, remains unknown. The II-III loop of Ca V 1.1 has no known structure but is known to contain a centrally located “critical domain” (∼residues 720-765) that is important for both tetrads and activation of calcium release. This region also appears to contain a binding site which likely causes the C terminus of the adapter-protein Stac3 to be close (∼3 nm) to the critical domain. Here, we have exploited our ability to recapitulate the coupling between Ca V 1.1 and RyR1 by expressing them, together with β1a, Stac3 and a junctophilin, in HEK293 cells, and using ECFP linked to the C terminus of Stac3 as a surrogate for the position of the II-III loop critical domain. FRET efficiency was measured between the ECFP and citrine fluorescent protein inserted at eight different RyR1 sites. Based on FRET efficiency, the II-III loop critical domain appears to be oriented toward the corner of the RyR1 cytoplasmic domain. Thus, it may account for the unidentified density connecting Ca V 1.1 to the RyR1 P1 domain, or that connecting to the P2 domain, both of which were revealed by electron tomography of muscle triad junctions in 2024 by Xu et al.
Heebner et al. (Sun,) studied this question.