Identification of a region of RyR1 that participates in allosteric coupling with the α_1S (Ca_v1.1) II-III loop

In skeletal muscle, excitation-contraction (EC) coupling and retrograde signaling are thought to result from direct interactions between the ryanodine receptor (RyR1) and the α₁ subunit of the dihydropyridine receptor (α_1S). Previous work has shown that the s53 region of α_1S (residues 720-765 in the II-III loop) and regions R10 (1635-2636) and R9 (2659-3720) of RyR1 are involved in this signaling. Using the yeast two-hybrid system, we here report an interaction between s53 and the sR16 region of RyR1 (1837-2168, within R10), whereas no interaction was seen using upstream residues of the α_1S II-III loop (s31, 666-709). The specificity of the s53-sR16 interaction was tested by using fragments of the cardiac RyR (RyR2) and DHPR (α_1C) that correspond to sR16 and s53, respectively. No interaction was observed for sR16 × c53 (α_1C 850-897), but weak interaction was occasionally observed for s53 × cR16 (RyR2 1817-2142). To test the functional significance of the s53 × sR16 interaction, we expressed in dyspedic myotubes a chimeric RyR (chimeraR16) in which sR16 was substituted for the corresponding region of RyR2. ChimeraR16 was found to mediate weak skeletal-type EC coupling. To test the necessity of sR16 sequence for coupling, we used "chimeraR16-rev," in which sR16 and a small upstream region of RyR1 were replaced by RyR2 sequence. ChimeraR16-rev did not differ from RyR1 in its ability to mediate EC coupling. Thus, interaction between residues 720-765 of α_1S and residues 1837-2168 of RyR1 appears to contribute to but is not essential for EC coupling in skeletal muscle.