Figure 3: Alcohol binding to ethanol-sensitized GLIC.

(a) Left, extracellular view of full transmembrane domain showing the sigma-A-averaged 2Fo–Fc electron density map (blue) contoured at 1.0 σ surrounding the refined position of ethanol (orange). Top right, equivalent density relative to contiguous transmembrane cavities (white) associated with one subunit interface. Bottom right, same representation for GLIC F14′A co-crystallized with 2-bromoethanol. The 2Fo–Fc electron density (blue) is overlaid with bromine-specific anomalous map (magenta) contoured at 5.0 σ. All maps are pictured within 2 Å of the modelled ligands. (b) Inter-subunit interface of F14′A plus ethanol, showing M1–M2 (dark grey) and M2–M3 (light grey) from neighbouring subunits, backbone and side-chain atoms for interfacial 14′ residue, and all amino acids within 4 Å of ethanol. Dashes indicate three possible hydrogen bonds. Residue N200 neighbours ethanol via its backbone carbonyl, and only its backbone is shown. (c) Proton response curves for GLIC wild-type (black), F14′A (orange) and F14′A-containing mutants I16′F (green), L17′F (yellow), N15′A (pink) and N15′A/E19′A (purple). Dashes indicate EC₁₀. (d) Sample traces showing EC₁₀ activations (H⁺) of GLIC F14′A, F14′A/I16′F and F14′A/N15′A in the absence and presence of 200 mM ethanol (E). Scale bars: 500 nA, 2 min. (e) Modulation by 200 mM ethanol of GLIC currents; n=5–16. (f) Modulation by 60 mM 2-bromoethanol, shown as in f; n=4–5. In f and g, bracket represents two-tailed unpaired t-test, not significant (NS); all other comparisons indicate analysis of variance, Dunnett’s test significance versus F14′A; ***P<0.001.