Organization of calcium channel β1a subunits in triad junctions in skeletal muscle

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 alpha(1S) subunit, the beta(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 beta(1a) subunits labeled with cyan fluorescent protein ( CFP) and/ or yellow fluorescent protein ( YFP). Expressed in dysgenic (alpha(1S)- null) myotubes, YFP-beta(1a)- CFP and CFP- beta(1a)- YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, beta(1a) does not appear to bind to other cellular proteins in the absence of alpha(1S). FRET efficiencies for these cytoplasmic beta(1a) subunits were similar to 6 - 7%, consistent with the idea that < 10 nm separates the N and C termini. After coexpression with unlabeled alpha(1S) ( in dysgenic or beta(1)- null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In beta(1)- null myotubes, FRET efficiencies of doubly labeled beta(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 beta(1a) ( CFP or YFP at the N or C terminus) were expressed in beta 1- null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent beta(1a) subunits are located > 10 nm from one another.