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 alpha1S subunit, the beta1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of beta1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (alpha1S-null) myotubes, YFP-beta1a-CFP and CFP-beta1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, beta1a does not appear to bind to other cellular proteins in the absence of alpha1S. FRET efficiencies for these cytoplasmic beta1a subunits were approximately 6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled alpha1S (in dysgenic or beta1-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In beta1-null myotubes, FRET efficiencies of doubly labeled beta1a 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 beta1a (CFP or YFP at the N or C terminus) were expressed in beta1-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent beta1a subunits are located >10 nm from one another.
Highlights
The dihydropyridine receptors (DHPRs) consists of a principal ␣1S subunit, which contains the ion-conducting pathway and voltage sensing structures, together with auxiliary 1a, ␣2␦-1, and ␥1 subunits
Presence of ␣1S, the 1a constructs are incorporated into DHPRs that are present in punctate regions of the plasma membrane that form junctions with the sarcoplasmic reticulum (SR)
In the absence of ␣1S, there were no foci of 1a indicative of binding to other cellular structures and these 1a subunits appeared to be freely diffusible in the cytoplasm
Summary
Generation of the Constructs Expressed in Myotubes—Fig. 1 illustrates the 1a constructs that were used for this study. To test for evoked contractions, the myotubes were placed in physiological saline (containing in mM: 140 NaCl, 1.5 KCl, 1 MgCl2, 2.5 CaCl2, 11 glucose, 10 HEPES, pH 7.4 with NaOH) and stimulated with 100-V, 15–30-ms pulses applied via a patch pipette that contained physiological saline and was positioned near the myotube Images of these myotubes were acquired at a rate of ϳ11 Hz. The contractions were quantified by measuring the movement of an identifiable portion of a myotube across the visual field. To measure the recovery of yellow fluorescence after photobleaching of YFP, myotubes were first imaged for cyan and yellow fluorescence as already described above Within this image, a smaller region-of-interest was designated for photobleaching of YFP, which was accomplished with repeated scans of non-attenuated 514 nm excitation. Images of cyan and yellow fluorescence were obtained as above every ϳ30 s, beginning immediately after, and continuing until ϳ400 s after, completion of the bleaching
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