Organization of calcium channel beta1a 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 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.
Leuranguer
Valérie
V
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1617, USA.
Papadopoulos
Symeon
S
Beam
Kurt G
KG
D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000378:metabolism; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007477:Ions; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000378:metabolism*; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; C492923:Cacnb1 protein, mouse; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; C485182:Cyan Fluorescent Protein; D018076:DNA, Complementary; D007477:Ions; D008164:Luminescent Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002118:Calcium