Accessibility of targeted DHPR sites to streptavidin and functional effects of binding on EC coupling

Abstract

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.

Figure 1.

Schematic illustration of the BAD-DHPR fusion constructs. Constructs are designated by the sites of attachment of BAD (red circle) and YFP (yellow oval). In the α_1S II–III loop constructs, α_1S residues 672–685 were replaced by the BAD. For the α_1S C terminus, BAD was attached after residue 1860, or attached after residue 1667, or placed after 1667 followed by α_1S 1668–1860 (YFP-α_1S(1667-BAD)1860).

Figure 2.

In nonfixed myotubes, the α_1S N terminus, the α_1S proximal II–III loop, and the N and C terminus of β_1a are accessible to streptavidin (A–D, respectively). Left panels illustrate the localization of the YFP-DHPR fusion proteins (indicated in green), center panels illustrate the localization of the bound streptavidin (red), and right panels show the overlay. Bars, 5 μm.

Figure 3.

Differential accessibility of α_1S C-terminal sites to streptavidin in nonfixed myotubes. (A) Myotubes expressing YFP-α_1S1860-BAD did not display binding of streptavidin that colocalized with loci of fluorescent DHPRs. (B) Myotubes expressing YFP-α_1S1667-BAD displayed incomplete colocalization between loci of DHPRs and streptavidin binding. The arrow indicates a cluster of DHPRs with substantial binding of streptavidin, and the arrowhead a cluster of DHPRS showing very little binding of streptavidin. (C) Nearly complete colocalization was observed for YFP-α_1S(1667-BAD)1860. Bars, 5 μm.

Figure 4.

Diffusion and binding of streptavidin injected into living myotubes. (A1) 1 h after streptavidin (SA) injection of a normal myotube, diffuse red fluorescence could be observed throughout the cell. (A2) In the absence of DHPR-BAD fusion proteins, injected streptavidin is rapidly released after saponin permeabilization. 1 h after SA injection of a normal myotube, saponin was applied and an image obtained ∼15 min later with laser intensity, detector/amplifier gains, and offset set to be the same as for the images in B. (B) A 1-h period was sufficient to allow binding of injected streptavidin to BAD-α_1S-YFP expressed in a dysgenic myotube. Nonbound streptavidin was released by saponin permeabilization. Bars, 5 μm. (C) Colocalization of red and yellow fluorescent puncta from injected streptavidin-rhodamine and YFP-β_1a-BAD in a β₁-null myotube. Note that BAD-β_1a-YFP streptavidin binding has not yet been confirmed by confocal imaging.

Figure 5.

Excitation-contraction coupling is abolished by streptavidin binding to the proximal portion of the α_1S II-III loop, but is unaffected when binding occurs at the α_1S N terminus or β_1a N or C terminus. Electrically evoked contractions are shown for intact β₁-null myotubes expressing YFP-β_1a-BAD or BAD-β_1a-YFP, or dysgenic myotubes expressing BAD-α_1S-YFP or α_1S(671-BAD-686)-YFP. Prior to streptavidin injection, myotubes responded to extracellular stimulation with robust contractions (top). 2–4 h after streptavidin injection, the same individual myotubes were tested for their responses to identical stimulation (bottom). Vertical scale, arbitrary units. For BAD-β_1a-YFP–expressing cells, streptavidin binding following injection has not yet been confirmed by confocal imaging.

Figure 6.

Effect of streptavidin binding to the proximal α_1S II–III loop on depolarization- and 4-CmC–induced Ca²⁺ transients. Representative responses are shown for normal myotubes (A) and SA-injected α_1S(671-BAD-686)-YFP–expressing myotubes (B) to a 5-s application of either 80 mM KCl (top) or 0.5 mM 4-CmC (bottom). ΔF/F scale bar = 0.4 or 0.6 for the KCl responses of the normal and SA-injected myotubes, respectively, and 1.0 or 0.6 for the 4-CmC responses of the normal and SA-injected myotubes, respectively. (C) Summary of peak ΔF/F amplitudes for KCl (top) and 4-CmC (bottom) responses of either normal myotubes (Norm) or SA-injected α_1S(671-BAD-686)-YFP– expressing myotubes (SA-inj).

Figure 7.

Streptavidin binding to the proximal portion of the α_1S II–III loop has little effect on calcium currents. (A) Currents were recorded from a dysgenic myotube expressing α_1S(671-BAD-686)-YFP that was injected with streptavidin-rhodamine >2 h before recording. The plot shows the I-V relationship for α_1S(671-BAD-686)-YFP currents following streptavidin injection. (B) The average amplitudes of calcium currents evoked by a 200-ms voltage step to +40 mV from α_1S(671-BAD-686)-YFP–expressing myotubes that were not (−SA, n = 4 cells) or were (+SA, n = 8 cells) injected with streptavidin.

Figure 8.

Possible mechanisms for how binding of streptavidin to the α_1S II-III loop could interfere with EC coupling. (A) During normal EC coupling, membrane depolarization induces a conformational change in the α_1S II–III loop, which causes the activation of RyR1 (indicated by “starburst”) and release of calcium. The inserted BAD (red circle) is proximal to the “critical domain” (pink) and does not interfere with these events. (B) Binding of streptavidin could globally distort α_1S such that the channel protein becomes nonfunctional. This possibility appears to be excluded since streptavidin binding did not affect the amplitude or gross voltage dependence of calcium currents. (C) Binding of streptavidin could interfere with the RyR1-activating conformational change of the II–III loop or another cytoplasmic portion of the DHPR.