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Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Aurora, CO 80045

Correspondence to Kurt G. Beam: kurt.beam{at}uchsc.edu

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane (PM) serves as a Ca²⁺ channel and as the voltage sensor for excitation–contraction (EC coupling), triggering Ca²⁺ release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. In addition to being functionally linked, these two proteins are also structurally linked to one another, but the identity of these links remains unknown. As an approach to address this issue, we have expressed DHPR _1S or ß_1a subunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the 60-kD streptavidin molecule had access to the ß_1a N and C termini and to the _1S N terminus and proximal II–III loop (residues 671–686). Steptavidin also had access to these sites after injection into living myotubes. However, sites of the _1S C terminus were either inaccessible or conditionally accessible in saponin- permeabilized myotubes, suggesting that these C-terminal regions may exist in conformations that are occluded by other proteins in PM/SR junction (e.g., RyR1). The binding of injected streptavidin to the ß_1a N or C terminus, or to the _1S N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal _1S II–III loop abolished such contractions, without affecting agonist-induced Ca²⁺ release via RyR1. Moreover, the block of EC coupling did not appear to result from global distortion of the DHPR and supports the hypothesis that conformational changes of the _1S II–III loop are necessary for EC coupling in skeletal muscle.

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				N. M. Lorenzon and K. G. Beam Accessibility of Targeted DHPR Sites to Streptavidin and Functional Effects of Binding on EC Coupling J. Cell Biol., October 8, 2007; 179(1): i3 - i3. [Full Text]

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