FIGURE 6.

R-mTFD-MPAB inhibits [³H]azietomidate photolabeling of β3Met-227 (βM1) but not α4Met-269 (αM1). A and B, ³H (●, ○) and pmol of PTH-derivatives (□) released during sequencing of subunit fragments beginning at β3Arg-216 and at α4Met-256 isolated by rpHPLC from EndoLys-C digests of subunits in the 58/62 kDa (A, β3/δ) or 72 kDa (B, α4) gel bands isolated by SDS-PAGE from α4β3δ GABA_ARs (110 pmol of muscimol sites per condition) photolabeled with 3.5 μm [³H]azietomidate in the absence (●, □) or presence (○) of 20 μm R-mTFD-MPAB. rpHPLC fractions 28 and 29 (A) and 25–28 (B) were sequenced. A, the primary sequence began at β3Arg-216 (I₀ = 14 pmol, both conditions), and the peak of ³H release in cycle 12 indicated labeling of β3Met-227 at 220 cpm/pmol in the absence and at 50 cpm/pmol in the presence of R-mTFD-MPAB. A secondary sequence was present beginning at β3Ala-280 (β3M3, ∼1 pmol). B, the fragment beginning at α4Met-256 (I₀ = 1 pmol) was present, along with fragments beginning at α4Val-313 (α4M3, ∼2.6 pmol) and α4Ser-238 (∼1 pmol). The peak of ³H release in cycle 14 was consistent with labeling of α4Met-269 (230 cpm/pmol) unaffected by R-mTFD-MPAB.