Ionotropic GABAA receptors (GABAARs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABAARs. We used Xenopus laevis oocytes to investigate the 6′ position in the second transmembrane region of GABAARs as a site influencing alcohol inhibition. We asked whether modification of the 6′ position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6′ position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6′ position. We verified a role for the 6′ position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABAARs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABAAR homolog, revealing that the 6′ position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6′ positions in both α2 and β2 GABAAR subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.
Ionotropic GABAA receptors (GABAARs) are ligand-gated ion channels that mediate the majority of inhibitory neurotransmission in the central nervous system (Farrant and Kaila, 2007). They belong to the Cys-loop superfamily, which includes glycine receptors (GlyRs), nicotinic acetylcholine receptors (nAChRs), and serotonin receptors. Functional receptors in this family are composed of five subunits, each containing an N-terminal extracellular domain, four transmembrane (TM) helices (TM1-TM4), and a cytoplasmic loop between TM3 and TM4. The ion pore is formed by the TM2 regions of all five subunits. Each TM2 region is a helix with residues numbered from the cytoplasmic (0′) to the extracellular (20′) end (Miller, 1989). The human genome encodes at least 16 different GABAAR subunits, with the most common stoichiometry being two α, two β, and one γ subunit. These subunits are arranged clockwise in the order αβαβγ, as viewed down the axis of the ion pore from the extracellular side (Trudell, 2002).
Behavioral and genetic studies implicate a variety of factors in manifestations of short- and long-term alcohol use (Harris et al., 2008). Particular attention has focused on GABAARs, which contribute to behavioral effects of ethanol, including tolerance, dependence, and withdrawal, are partially mediated through GABAARs (Chester and Cunningham, 2002; Boehm et al., 2004; Mehta and Ticku, 2005; Krystal et al., 2006; Lobo and Harris, 2008). Genetic studies in animals have provided evidence that the α1 and α2 subunits of GABAARs are required for specific behavioral actions of alcohol (Werner et al., 2006; Blednov et al., 2011). In addition, the GABRA2 gene, which encodes the α2 subunit, has been associated with alcohol use disorders in human populations (Enoch, 2008).
Molecular studies of alcohol action on GABAARs and related channels have suggested that alcohol acts at extracellular sites (Perkins et al., 2009), intracellular sites (Guzman et al., 2009), and transmembrane sites (Mihic et al., 1997; Mascia et al., 2000; Borghese et al., 2003; Crawford et al., 2007). Specifically, in GABAARs, residues α2(Ser270) and β1(Ser265) (both 15′ positions on TM2) and α2(Ala291) and β1(Met286) (two equivalent positions on TM3) are necessary for the potentiating effects of alcohol and are implicated in an alcohol interaction pocket between TM2 and TM3 (Mihic et al., 1997; Mascia et al., 2000). However, alcohol action is likely to be complex and may involve interaction with multiple sites (Howard et al., 2011b). Indeed, GABAARs, GlyRs, and nAChRs show evidence of inhibitory as well as potentiating effects of alcohol. In GABAARs, mutation of the TM2 potentiating sites in the mutant α2(S15′I)β1(S15′I) resulted in ethanol inhibition that was of the same magnitude as ethanol potentiation of the wild type (Mihic et al., 1997), suggesting that blockage of the enhancing site had unmasked an inhibitory mechanism. Likewise, in GlyRs, blockage of an engineered cysteine at the TM2 site α1(Ser15′) by covalent labeling with methanethiosulfonate (MTS) reagents unmasked an inhibitory action of ethanol (Crawford et al., 2007). In nAChRs, similar experiments provided evidence of both enhancing and inhibitory sites on the α2 subunit TM2 region (Borghese et al., 2003). These results suggest that the physiological effects of alcohol represent the summation of enhancing and inhibitory interactions.
Evidence supports the existence of an inhibitory site of alcohol action at the 6′ position in TM2 of GABAAR subunits. Substitution with tryptophan at residue Thr261 (Thr6′) in the GABAAR α2 subunit enhanced alcohol potentiation (Ueno at el., 2000), suggesting that the tryptophan moiety blocked an alcohol inhibitory site and unmasked additional enhancement by alcohol. Furthermore, cross-linking studies have provided evidence that the 6′ positions of GABAAR α and β subunits interact (Horenstein et al., 2001; Shan et al., 2002; Yang et al., 2007) and may contribute to a combined inhibitory site for anesthetics (Rosen et al., 2007). In this study, we used site-directed mutagenesis and MTS labeling of GABAARs expressed in Xenopus laevis oocytes to test the hypothesis that 6′ position residues in both α2β2- and α2β2γ2-containing GABAARs contribute to the inhibitory effects of alcohol.
Materials and Methods
Adult female X. laevis frogs were purchased from Xenopus Express, Inc. (Plant City, FL) or Nasco (Fort Atkinson, WI). GABA, methanol (MeOH), ethanol (EtOH), butanol (BuOH), hexanol (HeOH), and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO). MTS reagents (methyl and hexyl) were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada), and etomidate was purchased from Tocris Bioscience (Ellisville, MO). All chemicals used were of reagent grade.
Mutagenesis and Expression.
Human α2 and γ2s GABAAR subunits were contained in the pCIS2 vector, and the human β2 subunit was in the pBK-CMV vector. The desired tryptophan (α2 T261W), cysteine (α2 T261C and β2 T256C), or methionine (α2 T261M) mutations were produced by using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). In brief, complementary mutagenic primers were designed at the site of desired mutation. Polymerase chain reaction was performed by using primers and wild-type cDNA. All mutations were verified by using double-stranded DNA sequencing. X. laevis oocytes were manually isolated and treated with collagenase (type IA, 0.5 mg/ml) for 10 min and placed in sterile modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 NaHCO3, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, and 0.91 mM CaCl2, buffered to pH 7.5) supplemented with 10,000 units penicillin, 50 mg of gentamicin, 90 mg of theophylline, and 220 mg of sodium pyruvate per liter (incubation medium). Oocytes were injected with 50 nl of nuclease-free water containing wild-type or mutant α2β2 or α2β2γ2s at ratios of 1.5:1.5 or 1.5:1.5:4.5 ng, respectively. Oocytes were stored at 13°C in incubation medium until experiments were conducted.
Two-Electrode Voltage-Clamp Electrophysiology.
Recordings were performed as described previously (Borghese et al., 2003). In brief, GABA-induced currents were recorded at room temperature 2 to 7 days after cDNA injection. Oocytes were placed in a chamber and continuously bathed with ND96 buffer (96 mM NaCl, 2 mM KCl, 1 mM CaCl2·2H2O, 1 mM MgCl2·6H2O, and 5 mM HEPES, buffered to pH 7.5). Whole-cell voltage clamp was maintained at −70 mV via glass electrodes (1.5 MΩ) filled with 3 M KCl by using a Warner Instruments (Hamden, CT) model OC-725 oocyte clamp. All drugs were prepared immediately before bath perfusion. Methanol, ethanol, butanol, and GABA were diluted directly into ND96 buffer. Hexanol, etomidate, and stock MTS reagents were prepared in dimethyl sulfoxide and then diluted into ND96 buffer. Methyl methanethiosulfonate (MMTS) and hexyl methanethiosulfonate (HMTS) were applied at concentrations of 0.5 and 0.05 mM, respectively.
GABA response curves were determined by application of increasing GABA concentrations in 20- or 30-s intervals with 5- to 15-min washes. For all experiments, the maximal response was determined by applying 1 mM GABA (or 7 mM for the methionine mutant), and a concentration that stably produced 5% of maximal response (EC5) was used for all subsequent measurements. Modulation of GABA response by alcohols, anesthetics, or zinc was measured by a 1-min preapplication of modulator, followed by perfusion of modulator plus agonist for 30 s.
To investigate MTS reagent accessibility to the introduced cysteine residues, oocytes were perfused with MTS reagent for 1 min alone or followed by MTS plus EC5 or EC50 GABA for 30 s. Oocytes were then washed for 5 to 10 min in ND96 buffer. The current produced by EC5 GABA immediately before MTS perfusion was normalized to 100%. MTS effects were determined by the equation (Iafter/Ibefore − 1) × 100, where Ibefore and Iafter are current magnitudes induced by EC5 or EC50 agonist before and after application of the MTS reagent, respectively. MTS reagents [MMTS and 2-aminoethyl methanethiolsulfonate (MTSEA)] produced equivalent results when coapplied with EC5 as with EC50 GABA, indicating that over the 30-s coapplication period a similar number of channels became accessible to modification under both conditions. The effect of MMTS on alcohol modulation was determined by the aforementioned alcohol protocol, followed by treatment with MMTS plus EC5 or EC50 GABA, remeasurement of EC5, and repetition of the alcohol potentiation protocol. MTS reagents did not significantly alter baseline currents.
For experiments involving coapplication of MTS reagents with alcohols or anesthetics, EC5 or EC50 was determined as stated previously. Alcohol (methanol, ethanol, butanol, or hexanol) or etomidate was applied alone for 1 min, immediately followed by alcohol plus EC5 agonist plus MTS for 30 s. The effect of coapplication of alcohol/anesthetic and MTS was determined by using the aforementioned equation.
All values are represented as means ± S.E.M. from three or more independent experiments. Nonlinear regression analysis was performed to determine EC50 and Hill coefficient values from GABA concentration response curves by using Prism 5.01 (GraphPad Software, Inc., San Diego, CA). All data were normalized to maximal response and fitted to the equation IA = Imax/[1 + (EC50/A)n], where I is the current at GABA concentration A, Imax is the maximal current, EC50 is the GABA concentration that produces 50% of the maximal response, and n is the Hill coefficient. Statistical significance between wild-type and mutant receptors was determined by using two-tailed paired Student's t test or a one-way analysis of variance with the Bonferroni post hoc test.
A homology model of an α2β2γ2 GABAAR was generated by aligning the relevant sequences of human GABAAR subunits with the recently published sequence of an anion-selective glutamate receptor in the Cys-loop superfamily (GluClcryst; Protein Data Bank ID code 3RHW) (Hibbs and Gouaux, 2011). We first aligned each of the GABAAR α2, β2, and γ2s sequences with a single subunit in the homopentameric GluClcryst. Then we concatenated the GABAAR sequences to form five subunits in the order α2β2α2β2γ2 (Trudell, 2002). We used the Sequence Alignment module of Discovery Studio 2.5 (Accelrys, San Diego, CA) to produce an initial alignment. Then we manually aligned the sequences, based on the X-ray structure of GluClcryst, to assure that the residues important to the present studies were correctly displayed. We submitted that alignment to the Modeler module of Discovery Studio to build five similar models with the additional restriction to form and optimize the known Cys-loop disulfide bonds. The GABAAR sequences have inserts in several places compared with the sequence of GluCl. Typically these inserts are in loops that connect two β strands. We refined the structure of the loops in each of the five initial models. We calculated the total energy of each model by using the CHARMm force field and chose the one with the lowest (favorable) energy. To place the side chains of each amino acid in the best position relative to neighboring residues, we applied the Side Chain Refinement module of Discovery Studio on all residues of this model. To further improve the structure, this model was then optimized with CHARMm to a gradient of 0.0001 kcal/mol · Å in the presence of harmonic restraints on the backbone atoms of 10 kcal/Å2 and the Accelrys version of the CHARMm force field. Then we relaxed the structure with 100,000 1-fs steps of molecular dynamics at 300 K with the same harmonic restraints on the backbone atoms. Finally, we reoptimized the model as described above.
To model local structural changes associated with cysteine substitution or MMTS labeling, cysteine or methionine was substituted at the 6′ positions of both α subunits. Mutant models were then optimized with CHARMm as described above. The MMTS-labeled structure was approximated by replacing the Cγ atom of the substituted methionine residues with sulfur by using the Build Structure module. Images were produced by using the Persistence of Vision Raytracer (http://www.povray.org/).
The primary objective of this work was to determine whether the 6′ position in TM2 of α2 and β2 GABAAR subunits influences the inhibitory effects of alcohols on the function of the receptor. We hypothesized that the increased molecular volume contributed by tryptophan or methionine substitutions, or the labeling of an introduced cysteine with MMTS, would block any inhibitory effect of alcohols, as well as mimic the presence of alcohols by reducing receptor function.
Tryptophan Substitution in the α2 GABAAR 6′ Position Unmasks Potentiation by Short-Chain Alcohols.
To extend previous evidence of 6′ tryptophan substitutions blocking alcohol inhibition of α2β2 GABAARs (Ueno et al., 2000), we examined the effect of n-alcohols and volatile anesthetics on GABAARs of the same composition (α2β2) containing the tryptophan substitution α2(T6′W). Because GABAARs are implicated in immobilizing effects of n-alcohols and volatile anesthetics (Quinlan et al., 1998), we initially tested alcohol and anesthetic concentrations previously shown to produce 50% immobility (Alifimoff et al., 1989). Compared with wild-type receptors, tryptophan mutants showed greater enhancement by methanol and ethanol, but not by propanol, butanol, pentanol, hexanol, isoflurane, or chloroform (Fig. 1A). Consistent with previous findings (Ueno et al., 2000), tryptophan-mutant but not wild-type receptors were also sensitive to a lower concentration of ethanol (30 mM), approximately twice the legal threshold for intoxication (Harris et al., 2008), as well as an equivalently reduced concentration of methanol (89 mM) (Fig. 1, B–D). Also consistent with past studies (Ueno et al., 2000), the introduction of tryptophan at position 6′ did not significantly affect GABA responses (Table 1).
Most GABAARs in brain are heteromers of α, β, and γ subunits (Möhler, 2006); therefore, to probe a more physiologically relevant model system, we tested alcohol modulation of α2β2γ2s GABAARs. We confirmed incorporation of the γ subunit by first testing modulation by 1 μM zinc, which potently inhibits α2β2 but not α2β2γ2s GABAARs (Hosie et al., 2003; Trudell et al., 2008). Oocytes injected with GABAAR cDNA including the γ2s subunit exhibited less than 10% zinc inhibition (data not shown). In Fig. 2, sample traces show alcohol modulation of wild-type α2β2γ2s and mutant α2(T6′W)β2γ2s GABAARs. Compared with wild type, α2(T6′W)β2γ2s GABAARs showed greater enhancement by high concentrations of both methanol and ethanol (Fig. 2). These receptors were also sensitive to the lower concentration of methanol tested and showed a trend toward sensitivity to the lower concentration of ethanol (Fig. 2, B and C). We noted that the α2(T6′W) mutation in γ-containing receptors did enhance GABA sensitivity (Table 1), which was consistent with past evidence that the bulky tryptophan substitution at residues in the pore-lining TM2 helix can alter gating properties (Ueno et al., 2000).
MMTS Labeling Inhibits α2(T6′C)β2 GABAARs.
Cysteine mutagenesis and disulfide trapping are commonly used to probe protein structure/function relationships (Karlin and Akabas, 1998) and have been specifically applied to mimic anesthetic and alcohol interactions (Mascia et al., 2000). We predicted that an alcohol inhibition site should be accessible to small MTS reagents, and MTS labeling of an engineered cysteine residue in such a site should result in persistent inhibition. To test this prediction, we introduced cysteine at the 6′ position of the α2 GABAAR subunit, expressed wild-type or cysteine-mutant α2 subunits with β2, and characterized the resulting channels before and after treatment with MMTS.
The α2(T6′C)β2 mutant receptors were similar to wild type in maximal responses and GABA affinity (Table 1). Application and washout of 500 μM MMTS in the presence of EC5 GABA produced a significant and persistent decrease in GABA response in α2(T6′C)β2 but not wild-type receptors (Figs. 3, A and B). Application and washout of 500 μM MMTS in the presence of EC50 GABA produced similar results (data not shown). When applied in the absence of GABA, MMTS did not alter subsequent GABA-induced currents for wild-type or cysteine-mutant receptors (Fig. 3C). Consistent with previous findings in α1(T6′C)β2γ2 receptors (Xu and Akabas, 1996), we also observed persistent modulation, in the positive direction, of α2(T6′C)β2 receptors by the positively charged MTS reagent MTSEA (data not shown). Neither wild-type nor α2(T6′C)β2 mutant GABA-elicited currents were altered by a 2-min application of 10 mM DTT before treatment with MTS reagents (data not shown), indicating that neither receptor type underwent spontaneous cross-linking.
MMTS Labeling or Methionine Substitution at the α2 6′ Position Unmask Alcohol Potentiation.
We hypothesized that if MMTS labeling leads to occupation of a receptor site mediating alcohol inhibition, then MMTS should reduce alcohol inhibition. Previous studies have shown that blocking alcohol potentiation with MMTS reagents resulted in channels that were inhibited by alcohol, presumably due to the unmasking of inhibitory effects (Borghese et al., 2003; Crawford et al., 2007). Here, we predicted the opposite phenomenon: labeling the 6′ position with MMTS should reduce inhibitory binding and thereby augment net alcohol potentiation, in parallel to the tryptophan substitution results shown above. Indeed, treatment by MMTS robustly enhanced potentiation of α2(T6′C)β2 receptors by either methanol (Fig. 4A) or ethanol (Fig. 4B). We noted that cysteine substitution initially reduced short-chain alcohol potentiation relative to wild type, without altering potentiation by the anesthetic etomidate (Fig. 4C); MMTS treatment restored modulation to approximately wild-type levels (Fig. 4, A and B). There was no change in wild-type receptor modulation by either alcohol after MMTS treatment (Fig. 4, A and B).
Given that MMTS labeling may not have been complete, possibly accounting for the lack of change in alcohol effects relative to wild type, we noted that the structure of a methanethiolated cysteine residue (Fig. 4D) is similar to that of a methionine residue (Fig. 4E). Therefore, we expect a substituted methionine to duplicate the effects of complete MMTS labeling. Indeed, compared with wild type, α2(T6′M)β2 mutant receptors showed enhanced potentiation by methanol and ethanol, but not by hexanol or etomidate (Fig. 4F). The methionine mutation also reduced receptor sensitivity to GABA (Table 1), which is consistent with occupation of an inhibitory site by the bulkier residue.
Alcohols Prevent MMTS Modification of α2(T6′C)β2 GABAARs.
Our results indicate that the 6′ position mediates inhibition by both alcohols and MMTS, possibly via a shared binding site. We hypothesized that if alcohols and MMTS reagents indeed interact with a shared site, then coapplication with alcohols could prevent MMTS action at the 6′ position. Indeed, coapplication of an immobilizing concentration of methanol (590 mM) (Alifimoff et al., 1989) with MMTS and GABA prevented inhibition of receptor function (Fig. 5A). Conversely, an immobilizing concentration of butanol (11 mM) did not block the effects of MMTS (Fig. 5B); in fact, butanol concentrations as high as 33 mM failed to prevent inhibition by MMTS (data not shown). This result corresponds to our tryptophan mutant data indicating that inhibition via the 6′ position is specific to short-chain alcohols (Fig. 1A); however, we also observed competition between MMTS and the longer-chain alcohol hexanol (Fig. 5C), as discussed in more detail below.
To further substantiate a direct interaction with the 6′ position, we asked whether etomidate could compete with MMTS labeling. Etomidate is thought to bind at the lipid-facing α-β interface at the extracellular end of the transmembrane domain (Li et al., 2009), distal to the internal pore-facing 6′ positions; therefore, we expected it would not interfere with MMTS-induced persistent inhibition of GABA-activated currents. Indeed, whereas 1 μM etomidate modulated α2(T6′C)β2 receptors to an equivalent degree as wild type (Fig. 4C), coapplication of the anesthetic did not interfere with MMTS inhibition (Fig. 5D).
Hexyl Reagents May Interact Silently with the α2 6′ Position.
The changes we observed in alcohol modulation of tryptophan mutants (Fig. 1A) suggested that, whereas the 6′ position mediates inhibitory effects of methanol and ethanol, it does not influence inhibition by longer-chain alcohols. To test the accessibility of the 6′ position to longer-chain reagents, we treated wild-type and α2(T6′C)β2 GABAARs with HMTS. Indeed, HMTS applied alone (data not shown) or in the presence of agonist had no effect on GABA-induced currents in wild-type or mutant receptors (Fig. 6A).
To reconcile this result with our previous observation that hexanol competes with MMTS labeling (Fig. 5C), we hypothesized that hexanol and HMTS might interact with the 6′ position silently, that is, without affecting the function of the receptor. To test this hypothesis, we treated mutant GABAARs successively with HMTS and MMTS. After HMTS treatment, MMTS failed to produce persistent inhibition of GABA responses (Fig. 6B), suggesting that HMTS had covalently blocked the 6′ position. Wild-type responses were not significantly different from mutant responses in the presence of HMTS.
Cysteine Substituted in the β2 GABAAR 6′ Position Shows Evidence of Spontaneous Cross-Linking.
To investigate the contribution of β subunits to alcohol inhibition, we introduced cysteine at the 6′ position of the β2 subunit and coexpressed it with wild-type α2. Although the GABA EC50 for α2β2(T6′C) receptors was not different from wild type, the maximal response was reduced by more than 50% (Table 1). Successive applications of agonist caused escalating rundown, whereas perfusion with 10 mM DTT led to a 2-fold increase in agonist response (data not shown). We concluded that β2(T6′C) subunits formed spontaneous cross-links, most likely between adjacent subunits, which could be at least partially reversed by DTT treatment. We noted that adjacent β subunits are found only in αβ GABAARs, whereas in αβγ GABAARs the γ subunit separates the two β subunits (Trudell et al., 2008). These findings are consistent with previous reports in α1β1 GABAARs that adjacent β subunits cross-link via engineered Thr6′ cysteines in the closed state, whereas nonadjacent α subunits do not cross-link (Shan et al., 2002; Yang et al., 2007). Because of the formation of spontaneous disulfide bonds, we were unable to fully characterize alcohol and MTS reagent modulation of α2β2(T6′C) GABAARs.
MMTS Labeling Inhibits α2(T6′C)β2γ2s and α2β2(T6′C)γ2s GABAARs.
We also assessed MMTS effects on the more physiologically relevant γ-containing GABAARs (Möhler, 2006). Incorporation of the γ subunit was confirmed by an absence of zinc modulation as described above. In γ-containing GABAARs, cysteine substitution at the α2(Thr6′) position increased sensitivity to GABA. Conversely, similar to α2β2 receptors, treatment of α2(T6′C)β2γ2s receptors with MMTS in the presence of EC5 GABA reduced subsequent GABA-evoked currents (Fig. 7A, ■). MMTS treatment did not alter GABA responses in wild-type α2β2γ2s receptors (Fig. 7A, ○).
It is noteworthy that incorporating the γ2s subunit allowed us to characterize receptors containing the β2(T6′C) mutant subunit in more detail: these receptors showed no evidence of spontaneous cross-linking, presumably because γ replaces one of the adjacent β subunits and eliminates the proximal disulfide bond partner (Cromer et al., 2002; Trudell, 2002). Similar to α2(T6′C)-containing receptors, α2β2(T6′C)γ2s mutants exhibited enhanced sensitivity to GABA, whereas a single application of 500 μM MMTS and EC5 GABA produced a long-lasting decrease in GABA-evoked currents that was not observed in the wild type (Fig. 7B). Thus, occupation of the 6′ position in either the α or β GABAAR subunits inhibits channel function in physiologically relevant GABAAR subtypes.
Molecular Modeling Reveals Structural Consequences of 6′ Modification.
To characterize the structural characteristics of the 6′ position and effects of its substitution or labeling, we built a molecular model from the recent crystallographic structure of the GABAAR homolog GluCl from Caenorhabditis elegans (Hibbs and Gouaux, 2011) with energy optimization before and after 100,000 1-fs steps of molecular dynamics simulation. Modeling of the α2β2γ2s-containing GABAAR on GluCl (Fig. 8A) oriented the 6′ position residue extending into the channel pore approximately midway along the z-axis of the transmembrane domain; conversely, the 15′ residue was nearer the extracellular end of the transmembrane domain, facing toward the neighboring TM3 helix (Fig. 8B). Based on this molecular dynamics result, we built additional models replacing the 6′ position with cysteine or an MMTS-labeled cysteine. As expected, substitution with cysteine decreased side-chain volume and polarity in the proximal pore region, whereas MMTS labeling partially blocked the same region (Fig. 8C).
Our results indicate that alcohols have inhibitory as well as enhancing effects on GABAARs, mediated by the 6′ position in TM2. We demonstrate that a single amino acid substitution at this position is sufficient to alter the proposed inhibitory effect of short-chain alcohols on GABAARs. Replacement of the α2(Thr6′) position with the bulkier residues tryptophan or methionine selectively enhanced potentiation of receptor function by methanol and ethanol, which is consistent with blockage of an inhibitory site in this region. Conversely, substitution at the same site with cysteine, which is smaller than the wild-type threonine, selectively reduced potentiation by methanol or ethanol, which is consistent with the expansion of an inhibitory site to allow higher-affinity alcohol interactions. It is noteworthy that neither increased nor decreased volume at the 6′ position affected modulation by long-chain alcohols or etomidate, indicating that the inhibitory mechanism mediated by this region is restricted to short-chain alcohols. This result is reminiscent of nAChRs, in which modification of the 16′ position in TM2 selectively altered action of alcohols shorter than butanol, whereas modification of the 17′ position altered the action of alcohols longer than hexanol (Borghese et al., 2003).
In methionine-substituted α2β2 receptors, as well as cysteine-substituted α2β2γ2s receptors, GABA sensitivity varied in a manner consistent with occupation or evacuation of an inhibitory site, respectively; however, other substitutions showed opposite or insignificant changes in GABA potency, supporting past evidence that TM2 mutations may have a variety of effects on gating (Ueno et al., 2000), which may or may not be related to their effects on alcohol modulation.
Our experiments with MMTS provide more direct evidence for an inhibitory alcohol site at the 6′ position. In parallel to the well characterized potentiating sites at which MTS labeling enhanced GABAAR function (Mascia et al., 2000), MMTS labeling of engineered cysteines at the 6′ position of α2 or β2 subunits inhibited receptor function. Furthermore, whereas cysteine substitution at the 6′ position reduced short-chain alcohol potentiation (consistent with an enhanced inhibitory effect of these modulators), MMTS labeling of this residue restored alcohol potentiation to wild-type levels. Thus, in parallel to recent studies of GlyRs (Crawford et al., 2007) and nAChRs (Borghese et al., 2003), MTS reagents can be valuable tools for discriminating between multiple sites of alcohol interaction: substitution and labeling of our target residue revealed an inhibitory effect previously masked by the potentiating effects of alcohols at other sites in the same receptor. Furthermore, in parallel to recent work on the 15′ potentiating site (McCracken et al., 2010), we showed that coapplication of methanol blocked the inhibitory effect of MMTS on the 6′ cysteine mutant. This result is consistent with direct competition for a 6′-proximal inhibitory binding site, although we cannot exclude the possibility that allosteric modification of the receptor renders the 6′ site inaccessible to MMTS.
We were surprised to observe competition for MMTS labeling by hexanol, because our initial experiments indicated that the inhibitory effect of interaction at the 6′ position was restricted to short-chain alcohols, and treatment with a longer-chain MTS reagent (HMTS) did not alter GABA-induced currents. However, HMTS treatment did block subsequent labeling by MMTS, indicating that the 6′ position is indeed accessible to longer-chain reagents but that the interaction is silent (does not alter function). These findings highlight the need for caution in interpreting MTS reagent effects. Although a change in function after MTS treatment generally implies that a functionally relevant cysteine residue is accessible to the reagent (at least in some functional states of the protein), a lack of change in function does not necessarily imply inaccessibility; in some cases, MTS labeling may simply have no functional consequence (Karlin and Akabas, 1998). Furthermore, labeling of α2(T6′C)β2 receptors with the larger, positively charged MTS reagent MTSEA irreversibly potentiated GABA action both in our hands and in α1(T6′C)β2γ2s receptors (Xu and Akabas, 1996). Thus, specific chemical properties of a bound modulator or labeling agent may modify energetic pathways between open, closed, and desensitized states in complex ways; in the case of MTS reagents, properties such as charge and volume may determine whether receptor function is inhibited (MMTS), enhanced (MTSEA), or not changed (HMTS).
We also noted that MMTS labeling of 6′ position cysteines required coapplication of at least a low concentration of GABA. This labeling suggests that MMTS reactivity at the 6′ position is sensitive to conformational changes produced by channel gating, raising the possibility that binding of small molecules in this region may be selective for open or desensitized states of the receptor. It is noteworthy that MTSEA labeling at the 6′ position was observed in the presence and absence of GABA in our hands and others (Xu and Akabas, 1996), implicating either enhanced accessibility of this reagent to the closed state of the channel or its ability to act as an agonist and increase channel openings in the absence of GABA. Thus, the chemical properties of different MTS reagents may affect their sensitivity to protein conformational changes, sometimes with counterintuitive results: in this case, a larger reagent (MTSEA) could react where a smaller one (MMTS) did not.
Methionine substitution served as a valuable positive control for MMTS labeling in this study. The structural similarity between methionine and a methanethiolated cysteine was confirmed in a recent crystal structure of a protein labeled with MMTS, in which the labeled cysteine residue adopts a conformation interchangeable with that of a methionine residue (Brams et al., 2011). Previous work using methionine to mimic methanethiolation has allowed targeted “labeling” of engineered positions, ruling out nonspecific effects of MMTS on endogenous cysteines or other aspects of channel function (Howard et al., 2011a). This approach may be a valuable tool for controlling common variables in future MTS studies. It is noteworthy that whereas MMTS labeling restored alcohol potentiation approximately to wild-type levels, methionine substitution seemed to block alcohol inhibition even more effectively, enhancing alcohol potentiation relative to wild type. This result may reflect subtle differences between the structurally similar methionine and methanethiolated cysteine or indicate that MMTS labeling in our experimental protocol was not complete, whereas the methionine mutant necessarily contained the “labeled” residue in all α subunits in all receptors.
The relative participation of different GABAAR subunits in alcohol modulation is a topic of continuing debate (Lobo and Harris, 2008). An important contribution of our work is the consistency of the inhibitory site in a variety of GABAAR subunit arrangements. We initially investigated GABAARs containing only α2 and β2 subunits for the sake of consistency with previous studies (Ueno et al., 2000) and to simplify the number of different subunits contributing to the putative inhibitory site. Having tested a variety of α2β2 mutants, we extended this work by showing that tryptophan substitution at α2(Thr6′) also reduces alcohol potentiation of γ-containing receptors, which are the predominant population in brain (Möhler, 2006). Furthermore, although many structure/function studies on alcohol modulation have focused on GABAAR α subunits (Mascia et al., 2000), McCracken et al. (2010) recently demonstrated a critical role for the TM2 15′ position in the GABAAR β2 subunit in receptor potentiation by alcohols and anesthetics. We also showed conservation of the inhibitory effect of MMTS labeling at the 6′ position in α2 and β2 subunits, again in the context of γ-containing receptors. These results support the contribution of 6′ residues from multiple subunits, either to symmetrical inhibitory sites or a shared inhibitory region, in physiologically relevant GABAARs.
Although interpretation of structure/function data in GABAARs is challenging because of a lack of high-resolution structural data, the recent crystallographic determination of the structure of GluCl, a homolog of GABAARs from C. elegans, has provided a valuable new template for modeling (Hibbs and Gouaux, 2011). Our models of the α2β2γ2s GABAAR, the first to our knowledge based on the atomic-resolution eukaryotic homolog GluCl, revealed a pore-facing region dramatically influenced by modification of the α2 6′ position residue. Substitution at this position did not block the pore in any of the models, but did modify volume and polarity at one of the narrowest points in the conductive pathway. Conversely, the same models place the 15′ residue, which is well characterized as an alcohol potentiating site (Mihic et al., 1997), nearer the extracellular end of the transmembrane domain facing toward the neighboring TM3 helix. Our data support an inhibitory action of short-chain alcohols mediated by the pore-facing 6′ position, which may be independent of potentiating effects of alcohols at structurally distant positions, such as 15′. These results are consistent with previous studies supporting the existence of multiple interaction sites for alcohols on ligand-gated ion channels, in some cases with opposing functional consequences (Borghese et al., 2003; Crawford et al., 2007; Howard et al., 2011b). The biophysical basis for alcohol modulation continues to reveal startling complexity and will require further dissection at the molecular level to understand the interplay of low-affinity interactions that result in such dramatic effects on the brain and behavior.
Participated in research design: Johnson, Howard, and Harris.
Conducted experiments: Johnson.
Performed data analysis: Johnson, Howard, and Trudell.
Wrote or contributed to the writing of the manuscript: Johnson, Howard, Trudell, and Harris.
We thank Cecilia Borghese, Mandy McCracken, and Lindsay McCracken for technical support and many helpful discussions.
This work was supported by the National Institutes of Health National Institute of Alcohol Abuse and Alcoholism [Grants T32-AA007471, F32-AA019851, AA06399].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- GABAA receptor
- nicotinic acetylcholine receptor
- glycine receptor
- 2-aminoethyl methanethiolsulfonate
- methyl methanethiosulfonate
- propyl methanethiosulfonate
- hexyl methanethiosulfonate
- 5% of maximal response
- invertebrate glutamate-gated chloride channel receptor.
- Received September 21, 2011.
- Accepted November 8, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics