Effects of mutations to the human α7-AChR’s ECD or TMD domains on experimental competition curves. The binding reactions were incubated at 37°C for 24 or 48 h. All curves were best fitted with one-component Hill equations. For each construct, the fixed concentration of unbound [¹²⁵I]-α-BgTx was approximately equal to the corresponding toxin’s half-saturation concentration (Fig. 6 and Table 1). The number of independent competition assays contributing to each plotted curve is indicated in parentheses in the corresponding figure caption; errors were calculated only when the latter was >2. Error bars (±1 SEM) smaller than the size of the symbols were omitted. The concentration of unlabeled ligand (on the x axes) corresponds to the total (bound plus unbound) concentration. Under the low ligand-depletion conditions of our experiments, this concentration was deemed to be a good approximation for the concentration of unbound unlabeled ligand at equilibrium. (A) MLA. (B) Nicotine. (C) Carbamylcholine. The color code is the same for all panels. The effects of the S56T and S172T ECD mutations on the human–C. elegans α7-AChR–β-GluCl chimera were tested only for MLA. Half-competition and Hill-coefficient values are shown in Table 1.