Accessibility of Targeted DHPR Sites to Streptavidin and Functional Effects of Binding on EC Coupling

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 α_1S or β_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 β_1a N and C termini and to the α_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 α_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 β_1a N or C terminus, or to the α_1S N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal α_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 α_1S II–III loop are necessary for EC coupling in skeletal muscle.