S. John Mihic

Sites of alcohol and volatile anaesthetic action on GABA A and glycine receptors

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as γ-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABAA and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA ρ1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABAA and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.

Ethanol's molecular targets.

Ethanol produces a wide variety of behavioral and physiological effects in the body, but exactly how it acts to produce these effects is still poorly understood. Although ethanol was long believed to act nonspecifically through the disordering of lipids in cell membranes, proteins are at the core of most current theories of its mechanisms of action. Although ethanol affects various biochemical processes such as neurotransmitter release, enzyme function, and ion channel kinetics, we are only beginning to understand the specific molecular sites to which ethanol molecules bind to produce these myriad effects. For most effects of ethanol characterized thus far, it is unknown whether the protein whose function is being studied actually binds ethanol, or if alcohol is instead binding to another protein that then indirectly affects the functioning of the protein being studied. In this Review, we describe criteria that should be considered when identifying alcohol binding sites and highlight a number of proteins for which there exists considerable molecular-level evidence for distinct ethanol binding sites. For much of the 20th century, it was widely believed that ethanol exerts its effects on neuronal function in a nonspecific manner—perhaps through the disordering of membrane lipids. However, over the past two decades, evidence has mounted that ethanol instead produces its effects by altering the functioning of specific proteins through its interaction with a select few amino acids in those proteins. In this Review with 2 figures and 60 citations, we focus on proteins for which evidence for specific alcohol binding sites has been obtained, and we briefly describe and compare these ethanol receptors.

Inhaled drugs of abuse enhance serotonin-3 receptor function

Despite the prevalence of their use, little is currently known of the molecular mechanisms of action of inhaled drugs of abuse. Recent studies have shown effects on NMDA, GABA(A) and glycine receptors in vitro, suggesting that inhalants may exert at least some of their pharmacological effects on ligand-gated ion channels. Enhancement of serotonin-3 receptor function has been shown to play a role in the reinforcing properties of drugs of abuse. We tested the hypothesis that the commonly abused inhaled agents 1,1,1-trichloroethane, trichloroethylene, and toluene enhance serotonin-3 receptor function. All three inhalants significantly and reversibly potentiated, in a dose-dependent manner, serotonin-activated currents mediated by mouse serotonin-3A receptors expressed in Xenopus oocytes. Our findings add the serotonin-3 receptor to the growing list of molecular targets commonly affected by both inhalants and classic CNS depressants such as ethanol and the volatile anesthetics.

Tryptophan scanning mutagenesis in TM2 of the GABAA receptor α subunit: effects on channel gating and regulation by ethanol

Each residue in the second transmembrane segment (TM2) of the human GABAA receptor α2 subunit was individually mutated to tryptophan. The wild‐type or mutant α2 subunits were expressed with the wild‐type human GABAA receptor β2 subunit in Xenopus oocytes, and the effects of these mutations were investigated using two‐electrode voltage‐clamp recording. Four mutations (V257W, T262W, T265W and S270W) produced receptors which were active in the absence of agonist, and this spontaneous open channel activity was blocked by both picrotoxin and bicuculline, except in the α2(V257W)β2 mutant receptor, which was not sensitive to picrotoxin. Six mutations (V257W, V260W, T262W, T267W, S270W and A273W) enhanced the agonist sensitivity of the receptor, by 10–100 times compared with the wild‐type α2β2 receptor. Other mutations (T261W, V263W, L269W, I271W and S272W) had little or no effect on the apparent affinity of the receptor to GABA. Eight of the tryptophan mutations (R255, T256, F258, G259, L264, T265, M266 or T268) resulted in undetectable GABA‐induced currents. The S270W mutation eliminated potentiation of GABA by ethanol, whereas T261W markedly increased the action of ethanol. The T262W mutation produced direct activation (10% of maximal GABA response) by ethanol in the absence of GABA, while other mutations did not alter the action of ethanol significantly. These results are consistent with a unique role for S270 in the action of ethanol within the TM2 region, and with models of GABAA receptor channel function, in which specific residues within TM2 are critical for the regulation of channel gating (S270, L264), while other residues (L269, I271 and S272) have little effect on these functions and may be non‐critical structural residues.

Amino acid volume and hydropathy of a transmembrane site determine glycine and anesthetic sensitivity of glycine receptors

Two specific amino acid residues in transmembrane segments (TM) 2 and 3 are critical for the enhancement of glycine receptor (GlyR) function by volatile anesthetics. To determine which physicochemical characteristics of these sites determine their roles in anesthetic actions, an extensive series of single amino acid mutations at amino acid residue 288 (Ala-288) in TM3 of the α1 GlyR subunit was tested for modulation by volatile anesthetics. The mutations changed the apparent affinities of receptors for glycine; replacements with larger volumes and less hydropathy exhibited higher affinities for glycine. Potentiation by anesthetics was reduced by specific mutations at Ala-288. The molecular volume of the substituents was negatively correlated with the extent of potentiation by isoflurane, enflurane, and 1-chloro-1,2,2-trifluorocyclobutane, whereas there was no correlation between anesthetic enhancement and polarity, hydropathy, or hydrophilicity of substituents. In contrast to anesthetics, no correlation was found between the effects of the nonanesthetics 1,2-dichlorohexafluorocyclobutane or 2,3-dichlorooctafluorobutane and any physicochemical property of the substituent. These results suggest that the molecular volume and hydropathy of the amino acid at position 288 in TM3 regulate glycine and anesthetic sensitivity of the GlyR and that this residue might represent one determinant of an anesthetic binding site.

Antagonism of Inhalant and Volatile Anesthetic Enhancement of Glycine Receptor Function

Recent studies suggest that alcohols, volatile anesthetics, and inhaled drugs of abuse, which enhance γ-aminobutyric acid, type A, and glycine receptor-activated ion channel function, may share common or overlapping molecular sites of action on these receptors. To investigate this possibility, these compounds were applied singly and in combination to wild-type glycine α1 receptors expressed in Xenopus laevis oocytes. Data obtained from concentration-response curves of the volatile anesthetic enflurane constructed in the presence and absence of ethanol, chloroform, or toluene were consistent with competition for a common binding pocket on these receptors. A mutant glycine receptor, insensitive to the enhancing effects of ethanol but not anesthetics or inhalants, demonstrated antagonism of anesthetic and inhalant effects on this receptor. Although ethanol (25–200 mm) had no effect on its own in this receptor, it was able to inhibit reversibly the enhancing effect of enflurane, toluene, and chloroform in a concentration-dependent manner. These data suggest the existence of overlapping molecular sites of action for ethanol, inhalants, and volatile anesthetics on glycine receptors and illustrate the feasibility of pharmacological antagonism of the effects of volatile anesthetics.

Anesthetic and ethanol effects on spontaneously opening glycine receptor channels

Strychnine‐sensitive glycine receptors mediate inhibitory neurotransmission occurring in the brain stem and spinal cord. Alcohols, volatile anesthetics and inhaled drugs of abuse are positive allosteric modulators of glycine receptor function, normally enhancing function only in the presence of glycine. A complication in studying allosteric actions on ligand‐gated ion channels is in the dissection of their effects on neurotransmitter binding from their effects on channel opening. Mutation of an aspartate residue at position 97 to arginine in the glycine receptor α1 subunit simulated the effects of glycine binding, producing receptors that exhibited tonic channel opening in the absence of neurotransmitter; i.e. these receptors demonstrated a dissociation of channel opening from neurotransmitter binding. In these receptors, ethanol, enflurane, chloroform, halothane, 1,1,1‐trichloroethane and toluene elicited inward currents in the absence of glycine. We previously identified mutations on ligand‐gated ion channels that eliminate ethanol, anesthetic and inhalant actions (such as S267I on α1 glycine receptors). The double mutant (D97R and S267I) receptors were both constitutively active and resistant to the enhancing effects of ethanol and enflurane. These data demonstrate that ethanol and volatile anesthetics can affect glycine receptor channel opening independently of their effects on enhancing neurotransmitter binding.

Alcohol and benzodiazepines: Recent mechanistic studies

During the past three years, Medline identified approx. 14 000 articles with the textwords of ‘ethanol, alcohol, or alcoholism’ and 1400 with the textword ‘benzodiazepine’. Given this plethora of research activity, it is obvious that this brief review must not only focus on the recent literature but must also be restricted to a small subset of recent papers. We have elected to cover only mechanistic, mainly molecular, actions of ethanol and benzodiazepines on selected brain signaling systems. There are many recent review Ž articles covering areas omitted from this review see Luddens et al., 1995; Barnes, 1996; Crews et al., 1996; Phillips and Shen, 1996; Phillips et al., 1997; Tabakoff and Hoffman, 1996; Wilson, 1996; Diamond and Gordon, 1997; Herz, 1997; Miczek et al., 1997; Valenzuela . and Harris, 1997; Buck et al., 1998 . Areas of research progress include:

Ethanol potentiation of glycine receptors expressed in Xenopus oocytes antagonized by increased atmospheric pressure.

BACKGROUND Behavioral and biochemical studies indicate that exposure to 12 times normal atmospheric pressure (12 ATA) of helium-oxygen gas (heliox) is a direct, selective ethanol antagonist. The current study begins to test the hypothesis that ethanol acts by a common mechanism on ligand-gated ion channels by expanding previous hyperbaric investigations on gamma-aminobutyric acid type A (GABA(A)) receptors (GABA(A)Rs) at the biochemical level to alpha(1)glycine (GlyRs) expressed in Xenopus oocytes. METHODS Oocytes expressing wild-type alpha(1) homomeric GlyRs were voltage-clamped (-70 mV) and tested in the presence of glycine (EC(2)) +/- ethanol (50-200 mM) under 1 ATA control and 3 to 12 ATA heliox conditions. Glycine concentration response curves, strychnine/glycine interactions, and zinc (Zn2+) modulation of GlyR function was also tested. RESULTS Pressure reversibly antagonized the action of ethanol. The degree of antagonism increased as pressure increased. Pressure did not significantly alter the effects of glycine, strychnine, or Zn2+, indicating that ethanol antagonism by pressure cannot be attributed to alterations by pressure of normal GlyR function. The antagonism did not reflect tolerance to ethanol, receptor desensitization, or receptor rundown. CONCLUSION This is the first use of hyperbarics to investigate the mechanism of action of ethanol in recombinant receptors. The findings indicate that pressure directly and selectively antagonizes ethanol potentiation of alpha(1)GlyR function in a reversible and concentration- and pressure-dependent manner. The sensitivity of ethanol potentiation of GlyR function to pressure antagonism indicates that ethanol acts by a common, pressure-antagonism-sensitive mechanism in GlyRs and GABA(A)Rs. The findings also support the hypothesis that ethanol potentiation of GlyR function plays a role in mediating the sedative-hypnotic effects of ethanol.

Multiple sites of ethanol action in α1 and α2 glycine receptors suggested by sensitivity to pressure antagonism

The current study used an ethanol antagonist, increased atmospheric pressure, to test the hypothesis that ethanol acts on multiple sites in glycine receptors (GlyRs). The effects of 12 times normal atmospheric pressure of helium‐oxygen gas (pressure) on ethanol‐induced potentiation of GlyR function in Xenopus oocytes expressing human α1, α2 or the mutant α1(A52S) GlyRs were measured using two‐electrode voltage clamp. Pressure reversibly antagonized potentiation of glycine in α1 GlyR by 40–200 mm ethanol, but did not antagonize 10 and 25 mm ethanol in the same oocytes. In contrast, pressure did not significantly affect potentiation of glycine by 25–100 mm ethanol in α2 GlyRs, nor did pressure alter ethanol response in the A52S mutant. Pressure did not affect baseline receptor function or response to glycine in the absence of ethanol. These findings provide the first direct evidence for multiple sites of ethanol action in GlyRs. The sites can be differentiated on the basis of ethanol concentration, subunit and structural composition and sensitivities to pressure antagonism of ethanol. Parallel studies with butanol support this conclusion. The mutant α1(A52S) GlyR findings suggest that increased attention should be focused on the amino terminus as a potential target for ethanol action.