Structural basis for alcohol modulation of a pentameric ligand-gated ion channel

Abstract

Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the "linking tunnel." These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member.

Fig. 1.

Modulation by n-alcohols of GLIC currents in Xenopus laevis oocytes. Current traces show successive GLIC activations by pH 5.5 (≈pEC₁₀) in the presence and absence of (A) 590 mM methanol or (B) 570 μM hexanol. (Scale bars, 2 μA, 5 min). (C) Modulation by either short-chain n-alcohols (590 mM methanol, gray) or long-chain n-alcohols (36 μM octanol, black) was more pronounced at higher pH (lower level of activation). Errors are SEM, n = 2–30.

Fig. 2.

Profile of GLIC modulation by n-alcohols. (A) GLIC currents were potentiated by high concentrations of methanol or ethanol. Because it was not possible to collect data for complete concentration response relationships, curves represent linear regression fits. (B) GLIC currents were inhibited by n-alcohols larger than ethanol in a dose-dependent manner. Potency of inhibition by long-chain n-alcohols (propanol and larger) increased with chain length up to nonanol. Curves represent nonlinear regression fits as described in SI Methods. In A and B, errors are SEM, n = 2–18. (C) Region of GLIC (Protein Data Bank ID 3EAM) (25) transmembrane domain surrounding M2 residues previously implicated in alcohol modulation of eukaryotic pLGICs, shown as spheres: L(17′) (L241, yellow) and I(16′) (I240, green), homologous to the L263 “excitatory site” and L262 “inhibitory site” in M2 of the α2 nAChR (21); and N(15′) (N239, pink), homologous to S267 in the α1 GlyR (19). Position F(14′) (F238, orange), demonstrated in this study to strongly influence alcohol modulation of GLIC, is also shown. Cavities (black) neighboring the implicated residues were calculated using the Hollow script as described in SI Methods. Upper: Two GLIC subunits (gray, white) viewed from the plane of the membrane in the channel pore; cavity regions corresponding to the pore lumen are removed for clarity. Transmembrane helices M1–M4 are labeled in one subunit. Lower: Full pentameric channel transmembrane domain and cavities, viewed from the extracellular side. (D) Views as in C. Upper: Solvent-excluded surfaces surrounding cavities defined in C, with polar (blue), nonpolar (red), and intermediate (purple) regions colored by residue hydrophobicity. For clarity, M2 helices are not shown. Lower: Internal cavities as in C with intrasubunit (red) and intersubunit (blue) regions colored independently of linking tunnels and pore lumen (purple). Transmembrane helices are labeled in one subunit.

Fig. 3.

Effects of cysteine substitutions in M2 on n-alcohol modulation. (A) Modulation by 600 mM ethanol (Upper) or 11 mM butanol (Lower) of wild-type GLIC and 11 mutants. Channels exhibiting ethanol potentiation not significantly different from wild type are shown in black; mutants that ablated (13′, 16′; white) or enhanced (14′, 17′; gray) ethanol potentiation are colored independently (significance vs. wild-type, Dunnet's multiple comparison test, analysis of variance). (B) Sample pH response curves showing enhanced proton sensitivity of mutant A(10′)C (black circles) and reduced proton sensitivity of A(13′)C (white circles) and F(14′)C (gray circles, dotted curve) relative to wild type (crosses). Curves represent nonlinear regression fits as described in SI Methods. Dotted line represents 10% activation, at which alcohol modulation was measured. (C) Lack of correlation between changes in gating and ethanol potentiation for wild-type (crosses) and cysteine-substituted channels (circles) colored as in A. (D) Modulation of wild-type (black) and F(14′)C (gray) GLIC by a range of n-alcohols: 590 mM methanol, 600 mM ethanol, 86 mM propanol, 11 mM butanol, and 36 μM octanol. (E) Enhancement of potentiation in mutant F(14′)C (gray) relative to wild type (black) at a range of ethanol concentrations. In D and E, significance is vs. wild type, unpaired t test. (F) Potentiation by 200 mM ethanol of a variety of mutants substituted at position 14′ (significance vs. wild-type, Dunnet's multiple comparison test, analysis of variance). Errors are SEM, n = 2–17. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Labeling of cysteine mutants by MTS alcohol analogs. (A) GLIC current amplitudes for wild-type, C27A, and 10 M2 cysteine mutants after treatment with methyl MTS, relative to currents measured immediately before treatment (significance vs. modulation of wild-type GLIC, Dunnett's multiple comparison test, analysis of variance). (B) Reaction scheme showing labeling of cysteine-containing protein (1) by methyl MTS (2), yielding the methanethiolated protein (3) and a rapidly decomposing sulfinic acid (4). (C) Chemical structure of methionine in a protein. (D) Proton response curves for mutants in which L(17′) (gray, Left) or V(18′) (black, Right) was substituted with cysteine (circles), alanine (squares, dotted curve), or methionine (diamonds); curves fitted as in Fig. 3D. (E) Modulation by 600 mM ethanol (Upper) or 11 mM butanol (Lower) of GLIC mutants with cysteine or methionine substitutions at L(17′) (gray) or V(18′) (black) (significance of C vs. M mutants at each position, unpaired t test). Errors are SEM, n = 2–11. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Structural correlates of GLIC alcohol potentiation. (A) Wild-type (Left) and F(14′)A (Right) GLIC after 200-ns molecular dynamics simulations. Upper: M2 helices viewed from the extracellular side with kink position 9′ (green) and ethanol-enhancing position 14′ (orange) as spheres. Lower: Equivalent residues in the M2 helices of three subunits viewed from the channel pore. (B) Measurement of M2 helix kink angle over the course of wild-type (black) and F(14′)A (orange) simulations. Solid black line represents kink angle in GLIC crystal structure. (C) Region of GLIC surrounding key M2 positions after 200-ns simulation. Upper: Transmembrane helices from two subunits with positions 14′ (orange), 17′ (yellow), and 18′ (brown) on the proximal (gray) subunit shown as spheres. Average intrasubunit (red) and linking tunnel (purple) cavities associated with the proximal subunit were calculated using Fpocket as described in SI Methods. For clarity, only helices M2 and M3 of the distal (black) subunit are shown; in the proximal subunit, transmembrane helices M1–M4 are labeled. Lower: Equivalent view of mutant F(14′)A. (D) Average volumes across all five instances of each cavity type over the course of wild-type (black) and F(14′)A (orange) simulations. Upper: Similar pattern of intrasubunit cavities in both simulations. Lower: Larger, more variable linking tunnels in mutant F(14′)A relative to wild-type. In B and D, errors are SEM.