Junctional trafficking and restoration of retrograde signaling by the cytoplasmic RyR1 domain

Abstract

The type 1 ryanodine receptor (RyR1) in skeletal muscle is a homotetrameric protein that releases Ca²⁺ from the sarcoplasmic reticulum (SR) in response to an "orthograde" signal from the dihydropyridine receptor (DHPR) in the plasma membrane (PM). Additionally, a "retrograde" signal from RyR1 increases the amplitude of the Ca²⁺ current produced by Ca_V1.1, the principle subunit of the DHPR. This bidirectional signaling is thought to depend on physical links, of unknown identity, between the DHPR and RyR1. Here, we investigate whether the isolated cytoplasmic domain of RyR1 can interact structurally or functionally with Ca_V1.1 by producing an N-terminal construct (RyR1_1:4300) that lacks the C-terminal membrane domain. In Ca_V1.1-null (dysgenic) myotubes, RyR1_1:4300 is diffusely distributed, but in RyR1-null (dyspedic) myotubes it localizes in puncta at SR-PM junctions containing endogenous Ca_V1.1. Fluorescence recovery after photobleaching indicates that diffuse RyR1_1:4300 is mobile, whereas resistance to being washed out with a large-bore micropipette indicates that the punctate RyR1_1:4300 stably associates with PM-SR junctions. Strikingly, expression of RyR1_1:4300 in dyspedic myotubes causes an increased amplitude, and slowed activation, of Ca²⁺ current through Ca_V1.1, which is almost identical to the effects of full-length RyR1. Fast protein liquid chromatography indicates that ∼25% of RyR1_1:4300 in diluted cytosolic lysate of transfected tsA201 cells is present in complexes larger in size than the monomer, and intermolecular fluorescence resonance energy transfer implies that RyR1_1:4300 is significantly oligomerized within intact tsA201 cells and dyspedic myotubes. A large fraction of these oligomers may be homotetramers because freeze-fracture electron micrographs reveal that the frequency of particles arranged like DHPR tetrads is substantially increased by transfecting RyR-null myotubes with RyR1_1:4300 In summary, the RyR1 cytoplasmic domain, separated from its SR membrane anchor, retains a tendency toward oligomerization/tetramerization, binds to SR-PM junctions in myotubes only if Ca_V1.1 is also present and is fully functional in retrograde signaling to Ca_V1.1.