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J. Biol. Chem., Vol. 279, Issue 42, 44057-44064, October 15, 2004

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Metabolic Biotinylation as a Probe of Supramolecular Structure of the Triad Junction in Skeletal Muscle^*

Nancy M. Lorenzon, Claudia S. Haarmann, Ethan E. Norris, Symeon Papadopoulos, and Kurt G. Beam

From the Department of Biomedical Sciences, Anatomy Section, Colorado State University, Fort Collins, Colorado 80523

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 (60 kDa), myotubes were fixed, permeablized, and exposed to fluorescently labeled avidin. Upon expression in ₁-null or dysgenic (_1S-null) myotubes, punctate avidin fluorescence co-localized with the XFP puncta for BAD attached to the _1a N- or C-terminals, or the _1S N-terminal or II-III loop. However, BAD fused to the _1S 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 _1S C-terminal, which appears to be occluded by the presence of RyR1.

Received for publication, May 12, 2004 , and in revised form, July 20, 2004.

^* This work was supported by National Institutes of Health Grants NS24444 and AR44750 (to K. G. B.) and Deutsche Forschungsgemeinschaft Grant PA801/2-1 (to S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Physiologie 4220, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

To whom correspondence should be addressed: Dept. of Biomedical Sciences, Anatomy Section, 1617 Campus Delivery, Colorado State University, Fort Collins, CO 80523-1617. Tel.: 970-491-5277; Fax: 970-491-7907; E-mail: kbeam{at}lamar.colostate.edu.

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