Fig. 2. (A) Left panel: representative experiment of [¹²⁵I]α-BgTx binding to α7/5HT₃ receptor (white bar), ChimB (light gray bar), P193I (dark gray bar) and ChimB/P193I (black bar). Membrane fragments of each construction were incubated with 5 nM [¹²⁵I]α-BgTx for 2 h and samples were filtered and counted. Values indicated represent specific [¹²⁵I]α-BgTx binding (non-specific binding was determined in the presence of 1 mM nicotine). Data are means ± SE of triplicates. Right panel: representative experiment of specific (non-specific binding was determined in the presence of 1 mM nicotine) [³H]nicotine and [³H]epibatidine binding on ChimB (light gray bar) and ChimB/P193I (black bar). Membrane fragments were incubated with 100 nM [³H]nicotine or 10 nM [³H]epibatidine until equilibrium. Samples were filtered and counted. Data were normalized (a.u., arbitrary units) to ChimB values and are means ± SE of triplicates. (B) Representative experiment of [³H]epibatidine binding to the double mutant ChimB/P193I. Membrane fragments were incubated with various concentrations of [³H]epibatidine (0.5–20 nM) in the presence (filled squares) or absence (open circles) of 1 mM nicotine until equilibrium and samples were filtered and counted. Specific [³H]epibatidine is also indicated (filled circles). The inset shows a Scatchard plot of specific binding of [³H]epibatidine. Data were fitted to non-linear and linear least-squares analysis for the saturation and Scatchard plots, respectively, according to a single-site model. In this experiment, K_D = 11 ± 1 nM and B_max = 2.56 ± 0.07 nM. (C) Representative competition for [³H]epibatidine binding sites by α-BgTx for ChimB (filled circles) or ChimB/P193I (open circles) microchimeras. Membrane fragments were first incubated with various concentrations of α-BgTx (as indicated) until equilibrium, 10 nM [³H]epibatidine was then added for 1 h and samples were filtered and counted. Data were normalized to values determined in the absence of α-BgTx. Data points are means of duplicates of a representative experiment. Specific [³H]epibatidine binding was determined in the presence of 1 mM nicotine (filled square). Also indicated is [³H]epibatidine binding in the presence of 1 mM carbamylcholine (open square) for both mutants. Data were fitted to non-linear least-squares analysis according to a single-site inhibition model. Note that in this representative experiment, α-BgTx displaced at most 60% of bound [³H]epibatidine, whereas nicotine or carbamycholine displayed almost 100 and 91% of bound [³H]epibatidine, respectively.