The Cytoplasmic Domain of the RyR1 Foot is Sufficient for DHPR (Cav1.1) Organization into Tetrads

A RyR1 construct that lacks the channel forming C-terminal residues but includes the entire cytoplasmic foot region (YFP-RyR11:4300) forms a stable tetrameric structure (H. Bichraoui et al., abstract this meeting), colocalizes with DHPRs at SR/plasmalemma junctions and retrogradely enhances peak DHPR currents when expressed in dyspedic (RyR1 null) myotubes (A. Polster et al., abstract this meeting). We tested the interaction of the expressed RyR11:4300 with DHPRs in dyspedic myotubes by examining the DHPR disposition using freeze-fracture. Normally DHPRs are targeted to junctional sites in the absence of RyR, but their organization into tetrads and the arrangement of tetrads in ordered arrays is strictly dependent on their link to arrays of tetrameric RyRs. Thus the disposition of DHPRs in freeze-fracture images provides direct information both on DHPR/RyR interaction and on the arrangement of RyRs. In dyspedic cells DHPR clusters at peripheral couplings in a loose, completely random arrangement. Cells expressing full-length RyR1 show more tightly clustered DHPRs and the presence of complete (4 elements) and incomplete (2-3 elements) tetrads that are aligned in an orthogonal array. Cells expressing the truncated RyR11:4300 show small well identifiable DHPR foci with closely spaced particles. Some but not all foci show clear grouping of DHPRs into complete tetrads and/or tetrads composed of only three elements, but the tetrads are not aligned into a orthogonal array. We conclude that similar to intact RyR1, RyR11:4300 forms homo-tetramers which can link to DHPRs and organize them into tetrads, but that the cytoplasmic RyR1 foot differs from full-length RyR1 in that it does not form orthogonal arrays. The related positioning of RyR11:4300 and DHPRs is necessary for the retrograde interaction between the two.