The Cytoplasmic Foot of RyR1 without the Membrane Spanning Domain Targets Junctionally and Retrogradely Enhances DHPR L-Type Ca2+ Currents

Abstract

In skeletal muscle, RyR1 (5,037 residues) forms a homo-tetrameric Ca2+-release channel in the sarcoplasmic reticulum (SR), mediates excitation-contraction coupling in response to an orthograde signal from the DHPR in the plasma membrane, and retrogradely enhances L-type Ca2+ current via the DHPR. The RyR1 C-terminus forms the Ca2+ channel pore across the SR membrane and is believed to be important for inter-subunit interactions, whereas the bulk (∼85%) of the protein (the so-called foot) bridges the junctional, myoplasmic gap between the SR and plasma membranes. Here, we have examined the ability of the foot domain (residues 1-4300; RyR11:4300) to target junctionally and interact with the DHPR by expression of a cDNA encoding YFP-RyR11:4300. In dysgenic (α1S-null) myotubes which lack DHPRs, YFP-RyR11:4300 was diffusely distributed and, on the basis of photobleaching, freely mobile within the cytoplasm, consistent with the loss of membrane anchoring. However, after expression in dyspedic (RyR1 null) myotubes (which have DHPRs), much of YFP-RyR11:4300 was immobile within fluorescent foci near the myotube surface, suggestive of junctional targeting and binding to DHPRs. Junctional targeting was confirmed by partial co-localization of YFP-RyR11:4300 and CFP-labeled α1S after co-expression in dyspedic myotubes. Strikingly, YFP-RyR11:4300 was able to retrogradely enhance peak Ca2+ current in dyspedic myotubes from 1.6 pA/pF (control) to 6.7 pA/pF, similar to that after expression of full-length RyR1 (7.4 pA/pF). Thus, the isolated, cytoplasmic foot of RyR1 retains the ability to target junctionally and to interact functionally with the DHPR.