Figure 7. Identification of amino acids photolabeled by [¹²⁵I]-SADU-3-72 in the δ M2 segment in the desensitized Torpedo nAChR.

nAChR-rich membranes were labeled with [¹²⁵I]-SADU-3-72 in the presence of 400 μM Carb and in the absence (○, □) or presence 130 μM TCP (◆, ▼). As detailed in Experimental Procedures, the δ subunit was isolated from an 8% SDS-PAGE gel and digested ‘in-gel’ with V8 protease. The labeled fragment δV8-14K was isolated and further digested with trypsin. The fragment δT5K was then isolated from a small-pore Tricine SDS-PAGE gel. Shown are reversed-phase HPLC fractionation (A) and amino acid sequence analysis (B) of [¹²⁵I]-SADU-3-72-labeled proteolytic fragments δT-5K (A). The elution of peptides during HPLC was monitored by absorbance at 210 nm (solid line) and ¹²⁵I elution was quantified by γ counting of each fraction (○, ◆). B, ¹²⁵I (○, ◆ ) and PTH-amino acids (□, ▼) released during amino acid sequence analysis of ¹²⁵I peak (fractions 33–35; ○, 7,000 cpm; ◆, 3,000 cpm) from the HPLC purification of δT-5K. In each sample, the primary peptide detected began at δMet²⁵⁷ (□, I₀ = 9 ± 1 pmol, R = 91%; ▼, I₀ = 8 ± 1 pmol, R = 93%) and for the (◆) sample there were peaks of ¹²⁵I cpm release in cycles 2 and 9 corresponding to labeling of δSer²⁵⁸ (3 cpm/pmol) and δLeu²⁶⁵ (5 cpm/pmol).