Organization of Calcium Channel β_1a Subunits in Triad Junctions in Skeletal Muscle*

Abstract

In skeletal muscle, dihydropyridine receptors (DHPRs) in the plasma membrane interact with the type 1 ryanodine receptor (RyR1) at junctions with the sarcoplasmic reticulum. This interaction organizes junctional DHPRs into groups of four termed tetrads. In addition to the principle α_1S subunit, the β_1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of β_1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (α_1S-null) myotubes, YFP-β_1a-CFP and CFP-β_1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, β_1a does not appear to bind to other cellular proteins in the absence of α_1S. FRET efficiencies for these cytoplasmic β_1a subunits were ∼6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled α_1S (in dysgenic or β₁-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In β₁-null myotubes, FRET efficiencies of doubly labeled β_1a in puncta were similar to those of the same constructs diffusely distributed in the cytoplasm and appeared to arise intramolecularly, since no FRET was measured when mixtures of singly labeled β_1a (CFP or YFP at the N or C terminus) were expressed in β₁-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent β_1a subunits are located >10 nm from one another.