FIGURE 6.

[³H]TDBzl-etomidate labeling within αM2 in the presence of PCP or proadifen. ³H (○, ▴, ▵, ▿, and •) and PTH-amino acids (□) released during sequencing of α subunit fragments beginning at the amino terminus of αM2 are shown. The primary amino acid sequence is shown above the top panel. A and B, nAChR-rich membranes were photolabeled with 1 μm [³H]TDBzl-etomidate (75 μCi) on a preparative scale (10 mg of protein/condition) in +Carb (○), +Carb+PCP (▴), or PCP alone (▵). The fragments beginning at αMet-243 were purified by reversed-phase HPLC from EndoLys-C digests of αV8-20 (+Carb, I₀ = 7.5 pmol (A, □); +Carb+PCP, I₀ = 5.8 pmol; +PCP, I₀ = 7.3 pmol (B, □)). A, for nAChRs labeled in +Carb (○), the ³H releases in cycles 9, 10, and 13 indicated labeling of αLeu-251, αSer-252, and αVal-255 at 14, 13, and 8 cpm/pmol. Addition of PCP (▴) reduced the labeling of αLeu-251 to 1 cpm/pmol, whereas labeling of αSer-252 (18 cpm/pmol) was not inhibited. B, for nAChRs labeled in +PCP (▵), the ³H release in cycle 10 indicated labeling of αSer-252 at 250 cpm/pmol, i.e. at 20-fold higher efficiency than the labeling in +Carb (○). C, for nAChRs labeled in +proadifen (▿), the fragment beginning at αMet-243 was present at 2.3 pmol (□), and the ³H release in cycle 10 indicated labeling of αSer-252 at 225 cpm/pmol, which was 10-fold higher than the labeling in the resting state (•; from Fig. 5B).