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 Ca(2+) channel and as the voltage sensor for excitation-contraction (EC coupling), triggering Ca(2+) 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 alpha(1S) or beta(1a) subunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the approximately 60-kD streptavidin molecule had access to the beta(1a) N and C termini and to the alpha(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 alpha(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 beta(1a) N or C terminus, or to the alpha(1S) N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal alpha(1S) II-III loop abolished such contractions, without affecting agonist-induced Ca(2+) 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 alpha(1S) II-III loop are necessary for EC coupling in skeletal muscle.
Lorenzon
Nancy M
NM
Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Aurora, CO 80045, USA.
Beam
Kurt G
KG
D000818:Animals; D001665:Binding Sites; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D021122:Protein Subunits; D019837:Ryanodine Receptor Calcium Release Channel; C017071:biotin-binding proteins; D019809:Streptavidin