Metabolic biotinylation as a probe of supramolecular structure of the triad junction in skeletal muscle.
Excitation-contraction coupling in skeletal muscle involves conformational coupling between dihydropyridine receptors (DHPRs) in the plasma membrane and ryanodine receptors (RyRs) in the sarcoplasmic reticulum. However, it remains uncertain what regions, if any, of the two proteins interact with one another. Toward this end, it would be valuable to know the spatial interrelationships of DHPRs and RyRs within plasma membrane/sarcoplasmic reticulum junctions. Here we describe a new approach based on metabolic incorporation of biotin into targeted sites of the DHPR. To accomplish this, cDNAs were constructed with a biotin acceptor domain (BAD) fused to selected sites of the DHPR, with fluorescent protein (XFP) attached at a second site. All of the BAD-tagged constructs properly targeted to junctions (as indicted by small puncta of XFP) and were functional for excitation-contraction coupling. To determine whether the introduced BAD was biotinylated and accessible to avidin (approximately 60 kDa), myotubes were fixed, permeablized, and exposed to fluorescently labeled avidin. Upon expression in beta1-null or dysgenic (alpha1S-null) myotubes, punctate avidin fluorescence co-localized with the XFP puncta for BAD attached to the beta1a N- or C-terminals, or the alpha1S N-terminal or II-III loop. However, BAD fused to the alpha1S C-terminal was inaccessible to avidin in dysgenic myotubes (containing RyR1). In contrast, this site was accessible to avidin when the identical construct was expressed in dyspedic myotubes lacking RyR1. These results indicate that avidin has access to a number of sites of the DHPR within fully assembled (RyR1-containing) junctions, but not to the alpha1S C-terminal, which appears to be occluded by the presence of RyR1.
Department of Biomedical Sciences, Anatomy Section, Colorado State University, Fort Collins 80523, USA.
Nancy M
NM
Lorenzon
Claudia S
CS
Haarmann
Ethan E
EE
Norris
Symeon
S
Papadopoulos
Kurt G
KG
Beam
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D019921:Biotinylation; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology*; D008164:Luminescent Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000502:physiology*
D001426:Bacterial Proteins; D020746:Calcium Channels, L-Type; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C054562:yellow fluorescent protein, Bacteria