Fig. 3. (A) Competition binding curves for either [¹²⁵I]α-BgTx (broken curves; open symbols) or [³H]epibatidine (solid curves; filled symbols) binding sites by α-BgTx to α7/5HT₃ (open triangles), ChimB (filled and open circles) and G152K/P193I (filled triangles). For [¹²⁵I]α-BgTx binding, membrane fragments of α7/5HT₃ or ChimB were incubated with various concentrations of α-BgTx for 2 h, 2.5 nM [¹²⁵I]α-BgTx was added for 5 min (initial velocity conditions) and samples were filtered and counted. For [³H]epibatidine binding, the protocol given in the caption to Figure 2C was used. Data were normalized to values determined in the absence of α-BgTx. Non-specific binding was determined in the presence of 100 µM α-BgTx. Data are means ± SE of at least three independent experiments performed in duplicate. For clarity, standard errors were omitted for [¹²⁵I]α-BgTx binding for ChimB and α7/5HT₃ data. (B) Representative whole-cell patch–clamp traces of inhibition of ACh-induced currents by α-BgTx. Upper panel: application of 1 mM ACh for 3 s to a cell expressing α7/5HT₃ receptors induced an inward current (dark trace). Preincubation of the same cell with 20 nM α-BgTx for 10 min inhibited 1 mM ACh-induced current strongly (light trace). Lower panel: same protocol as in the upper panel but with the G152K/P193I double mutant. In this representative experiment, preincubation with 100 nM α-BgTx for 10 min did not inhibit 1 mM ACh-induced current. (C) Mutational scanning of α-BgTx affinity binding for the loop B region. The drawing on the right is a schematic representation of the double mutant series (see caption to Figure 1 for explanation). The α4 sequence (black box in the schematic representation) is shown in bold underlined type, while α7 is represented in light-face type. Data are means ± SE of at least two experiments (see Table I). The asterisk represents values that are statistically different from α7/5HT₃ using an unpaired Student’s t-test (p < 0.025).