Abstract
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.
Highlights
In skeletal muscle, two major proteins involved in excitationcontraction (EC)1 coupling are the dihydropyridine receptor (DHPR), a voltage-gated calcium channel in the plasma mem
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 ␣1S C-terminal, which appears to be occluded by the presence of RyR1
The preceding paper (8) described a new approach in which the fluorescent resonance energy transfer efficiency of a cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) tandem was used as an indirect indicator of sites of possible proximity between the DHPR and ryanodine receptors (RyRs)
Summary
Biotin Acceptor Domain—The BAD was extracted from the PinPoint Xa-1 expression vector (Promega, Madison, WI) containing the sequence encoding the PSTCD (Propionibacterium shermanii transcarboxylase domain). The 219-base pair (bp) fragment yielded by KpnI digestion of this modified PinPoint plasmid was inserted into KpnI-digested GFP-␣1Slong and GFP-␣1Sshort, which were constructed essentially as described by Papadopoulos et al (8) for YFP-␣1Slong and YFP-␣1Sshort In these two constructs, the rabbit ␣1S sequence (3) was terminated, respectively, after either amino acid 1860 or 1667 (fulllength ␣1S has 1873 residues). A 6-residue linker connected the 97-residue BAD to the N-terminal of ␣1S(III–IV)-YFP This construct was co-expressed with the plasmid ␣1S(I–II) (8). The ␣1S(I–II) coding sequence was isolated from a ␣1S(I–II) plasmid by restriction cuts with NheI and BamHI These two fragments were co-ligated to obtain ␣1S(I–II)-BAD-(III–IV)-YFP. GFP-BAD—Using PCR mutagenesis, a KpnI restriction site was introduced into the PSTCD sequence of the PinPoint Xa-1 plasmid directly before amino acid Glu (by inserting GGGGTACC-5Ј to nucleotide G157).
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