Fig. 2: Nicotine binding site.

a Left, the chemical structure of nicotine and the nicotine (cyan sticks) overlaid with its corresponding EM density (Blue mesh). Right, the top view of α6β4 structure bound with nicotine. b Loop A-F forming the nicotine binding pocket. Loop A-F are shown as different colors. Nicotine is showed as green spheres. c Interaction of nicotine in binding pocket. The key residues for interaction are showed as sticks. The nicotine is showed as sticks and spheres. The potential hydrogen bonds are represented as dashed lines. d Binding stability of nicotine in molecular dynamics simulations. Binding of nicotine measured by the distances from the hydrogen on the nitrogen of pyrrolidine ring to the carbonyl of W149 (blue line), and from the nitrogen of pyrrolidine ring to the carbonyl of L121 (red line). e Comparison of nicotine binding pocket in α6β4 and α3β4 receptors. The α3β4 receptor is colored as gray. The key residues for interaction in α6β4 and α3β4 receptors are represented by sticks and labeled. f Comparison of nicotine binding pocket in α6β4 and α4β2 receptors. The α4β2 receptor is colored as gray. The key residues for interaction in α6β4 and α4β2 receptors are represented by sticks and labeled. g Whole-cell concentration-response relationship of relative nicotine-evoked current amplitude comparing α6β4^WT and α6β4^L121F in two-electrode voltage clamp experiment. The black and blue curves represent α6β4^WT and α6β4^L121F, respectively. α6β4^WT EC₅₀ = 24.6 ± 1.5 μM (mean ± S.E.; 95% CI: 20.3–28.8 μM; n = 4, data from several experiments were pooled and each data point represents the average of 4 cells ±S.E.). α6β4^L121F EC₅₀ = 2.2 ± 0.4 μM (mean ± S.E.; 95% CI: 1.1–3.3 μM; n = 4, data from several experiments were pooled and each data point represents the average of 4 cells ± S.E.).