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Background: N-methyl-D-aspartate (NMDA) receptors regulate synaptic plasticity and modulate a wide variety of behaviors. Mammalian NMDA receptors are inhibited by ethanol (EtOH) even at low concentrations. In mice, the F639A mutation in transmembrane domain (TMD) 3 of the NR1 subunit reduces EtOH sensitivity of the receptor and, in some paradigms, reduces behavioral EtOH sensitivity and increases EtOH consumption. We tested the fly equivalent of the F639A and K544Q mutations for effects on EtOH sensitivity. Drosophila shows a high degree of behavioral and mechanistic conservation in its responses to EtOH., Methods: Homologous recombination and CRISPR/Cas9 genome editing were used to generate amino acid changes in the Drosophila NMDAR1 gene, yielding F654A and K558Q alleles. Animals were tested for the degree of EtOH sensitivity, the ability to acquire tolerance to EtOH, EtOH drinking preference, circadian rhythmicity, learning, and locomotor defects., Results: We observed that mutating the NMDAR1 channel also reduces EtOH sensitivity in adult flies. However, in flies, it was the K558Q mutation (orthologous to K544Q in mice) that reduces EtOH sensitivity in a recovery-from-sedation assay. The effects of the F654A mutation (orthologous to F639A in mice) were substantially different in flies than in mammals. In flies, F654A mutation produces phenotypes opposite those in mammals. In flies, the mutant allele is homozygous viable, does not seem to affect health, and increases EtOH sensitivity. Both mutations increased feeding but did not alter the animal's preference for 5% EtOH food. F654A depressed circadian rhythmicity and the capacity of males to court, but it did not depress the capacity for associative learning. K554Q, on the other hand, has little effect on circadian rhythmicity, only slightly suppresses male courtship, and is a strong learning mutant., Conclusions: Mutations in TMD 3 and in the extracellular-vestibule calcium-binding site of the NR1 NMDA subunit affect EtOH sensitivity in Drosophila., (© 2019 by the Research Society on Alcoholism.)

Ketogenesis is a metabolic process wherein ketone bodies are produced from the breakdown of fatty acids. In humans, fatty acid catabolism results in the production of acetyl-CoA which can then be used to synthesize three ketone bodies: acetoacetate, acetone, and β-hydroxybutyrate. Ketogenesis occurs at a higher rate in situations of low blood glucose, such as during fasting, heavy alcohol consumption, and in situations of low insulin, as well as in individuals who follow a 'ketogenic diet' consisting of low carbohydrate and high fat intake. This diet has various therapeutic indications, including reduction of seizure likelihood in epileptic patients and alcohol withdrawal syndrome. However, the mechanisms underlying these therapeutic benefits are still unclear, with studies suggesting various mechanisms such as a shift in energy production in the brain, effects on neurotransmitter production, or effects on various protein targets. Two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes was used to investigate the actions of ketone bodies on three ionotropic receptors: GABA A , glycine, and NMDA receptors. While physiologically-relevant concentrations of acetone have little effect on inhibitory GABA or glycine receptors, β-hydroxybutyrate inhibits the effects of agonists of these receptors at concentrations achieved in vivo. Additionally, both acetone and β-hydroxybutyrate act as inhibitors of glutamate at the excitatory NMDA receptor. Due to the role of hyperexcitability in the pathogenesis of epilepsy and alcohol withdrawal, the inhibitory actions of acetone and β-hydroxybutyrate at NMDA receptors may underlie the therapeutic benefit of a ketogenic diet for these disorders., (Copyright © 2018. Published by Elsevier Ltd.)

Alcohol is a widely used and abused substance. A major unresolved issue in the alcohol research field is determining which of the many alcohol target proteins identified to date is responsible for shaping each specific alcohol-related behavior. The large-conductance, calcium- and voltage-activated potassium channel (BK channel) is a conserved target of ethanol. Genetic manipulation of the highly conserved BK α channel influences alcohol-related behaviors across phylogenetically diverse species that include worm, fly, mouse, and man. A pharmacological tool that prevents alcohol's action at a single target, like the BK channel, would complement genetic approaches in the quest to define the behavioral consequences of alcohol at each target. To identify agents that specifically modulate the action of ethanol at the BK channel, we executed a high-throughput phagemid-display screen in combination with a Caenorhabditis elegans behavioral genetics assay. This screen selected a novel nonapeptide, LS10, which moderated acute ethanol intoxication in a BK channel-humanized C. elegans strain without altering basal behavior. LS10's action in vivo was dependent upon BK channel functional activity. Single-channel electrophysiological recordings in vitro showed that preincubation with a submicromolar concentration of LS10 restricted ethanol-induced changes in human BK α channel gating. In contrast, no substantial changes in basal human BK α channel function were observed after LS10 application. The results obtained with the LS10 peptide provide proof-of-concept evidence that a combined phagemid-display/behavioral genetics screening approach can provide novel tools for understanding the action of alcohol at the BK channel and how this, in turn, exerts influence over central nervous system function., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Benzodiazepines are positive allosteric modulators of the GABA A receptor (GABA A R), acting at the α-γ subunit interface to enhance GABA A R function. GABA or benzodiazepine binding induces distinct conformational changes in the GABA A R. The molecular rearrangements in the GABA A R following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABA A R, we identified electrostatic interactions between specific amino acids at the α-γ subunit interface that were broken by, or formed after, benzodiazepine binding. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes, we investigated these interactions by substituting one or both amino acids of each potential pair. We found that Lys 104 in the α 1 subunit forms an electrostatic bond with Asp 75 of the γ 2 subunit after benzodiazepine binding and that this bond stabilizes the positively modified state of the receptor. Substitution of these two residues to cysteine and subsequent covalent linkage between them increased the receptor's sensitivity to low GABA concentrations and decreased its response to benzodiazepines, producing a GABA A R that resembles a benzodiazepine-bound WT GABA A R. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The α 1 Lys 104 and γ 2 Asp 75 interaction did not play a role in ethanol or neurosteroid modulation of GABA A R, suggesting that different modulators induce different conformational changes in the receptor. These findings may help explain the additive or synergistic effects of modulators acting at the GABA A R., (© 2018 Pflanz et al.)

The amino acid taurine is an endogenous ligand acting on glycine receptors (GlyRs), which is released by astrocytes in many brain regions, such as the nucleus accumbens and prefrontal cortex. Taurine is a partial agonist with an efficacy significantly lower than that of glycine. Allosteric modulators such as ethanol and isoflurane produce leftward shifts of glycine concentration-response curves but have no effects at saturating glycine concentrations. In contrast, in whole-cell electrophysiology studies these modulators increase the effects of saturating taurine concentrations. A number of possible mechanisms may explain these enhancing effects, including modulator effects on conductance, channel open times, or channel closed times. We used outside-out patch-clamp single channel electrophysiology to investigate the mechanism of action of 200 mM ethanol and 0.55 mM isoflurane in enhancing the effects of a saturating concentration of taurine. Neither modulator enhanced taurine-mediated conductance. Isoflurane increased the probability of channel opening. Isoflurane also increased the lifetimes of the two shortest open dwell times while both agents decreased the likelihood of occurrence of the longest-lived intracluster channel-closing events. The mechanism of enhancement of GlyR functioning by these modulators is dependent on the efficacy of the agonist activating the receptor and the concentration of agonist tested., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

The glycine receptor is a pentameric ligand-gated ion channel that is involved in fast inhibitory neurotransmission in the central nervous system. Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nanomolar to low-micromolar concentrations and inhibiting its effects at higher concentrations. Low-nanomolar concentrations of contaminating zinc in electrophysiological buffers are capable of synergistically enhancing receptor modulation by other compounds, such as ethanol. This suggests that, unless accounted for, previous studies of glycine receptor modulation were measuring the effects of modulator plus comodulation by zinc on receptor function. Since zinc is present in vivo at a variety of concentrations, it will influence glycine receptor modulation by other pharmacologic agents. We investigated the utility of previously described "zinc-enhancement-insensitive" α 1 glycine receptor mutants D80A, D80G, and W170S to probe for interactions between zinc and other allosteric modulators at the glycine receptor. We found that only the W170S mutation conferred complete abolishment of zinc enhancement across a variety of agonist and zinc concentrations. Using α 1 W170S receptors, we established that, in addition to ethanol, zinc interacts with inhalants, but not volatile anesthetics, to synergistically enhance channel function. Additionally, we determined that this interaction is abolished at higher zinc concentrations when receptor-enhancing binding sites are saturated, suggesting a mechanism by which modulators such as ethanol and inhalants are capable of increasing receptor affinity for zinc, in addition to enhancing channel function on their own., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Partial agonists have lower efficacies than compounds considered 'full agonists', eliciting submaximal responses even at saturating concentrations. Taurine is a partial agonist at the glycine receptor (GlyR), a member of the cys-loop ligand-gated ion channel superfamily. The molecular mechanisms responsible for agonism are not fully understood but evidence suggests that efficacy at these receptors is determined by conformational changes that occur early in the process of receptor activation. We previously identified a residue located near the human α1 glycine binding site (aspartate-97; D97) that, when mutated to arginine (D97R), results in GlyR channels opening spontaneously with a high open probability, mimicking the effects of saturating glycine concentrations on wildtype GlyR. This D97 residue is hypothesized to form an electrostatic interaction with arginine-119 on an adjacent subunit, stabilizing the channel in a shut state. Here we demonstrate that the disruption of this putative bond increases the efficacy of partial agonists including taurine, as well as two other β-amino acid partial agonists, β-aminobutyric acid (β-ABA) and β-aminoisobutyric acid (β-AIBA). Even the subtle charge-conserving mutation of D97 to glutamate (D97E) markedly affects partial agonist efficacy. Mutation to the neutral alanine residue in the D97A mutant mimics the effects seen with D97R, indicating that charge repulsion does not significantly affect these findings. Our findings suggest that the determination of efficacy following ligand binding to the glycine receptor may involve the disruption of an intersubunit electrostatic interaction occurring near the agonist binding site., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Large conductance calcium-activated (BK) channels are broadly expressed in neurons and muscle where they modulate cellular activity. Decades of research support an interest in pharmaceutical applications for modulating BK channel function. Here we report a novel BK channel-targeted peptide with functional activity in vitro and in vivo. This 9-amino acid peptide, LS3, has a unique action, suppressing channel gating rather than blocking the pore of heterologously expressed human BK channels. With an IC 50 in the high picomolar range, the apparent affinity is higher than known high affinity BK channel toxins. LS3 suppresses locomotor activity via a BK channel-specific mechanism in wild-type or BK channel-humanized Caenorhabditis elegans. Topical application on the dural surface of the auditory midbrain in mouse suppresses sound evoked neural activity, similar to a well-characterized pore blocker of the BK channel. Moreover, this novel ion channel-targeted peptide rapidly crosses the BBB after systemic delivery to modulate auditory processing. Thus, a potent BK channel peptide modulator is open to neurological applications, such as preventing audiogenic seizures that originate in the auditory midbrain., Competing Interests: The authors declare no competing financial interests.

The glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (α1, α2, α3, and β) co-assemble to form pentameric channel proteins as either α homomers or αβ heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the α1β glycine receptor. This involved a panning procedure in which the phage library initially underwent subtractive panning against Human Embryonic Kidney (HEK) 293 cells expressing alternative glycine receptor subtypes before panning the remaining library over HEK 293 cells expressing the target, the α1β glycine receptor. Peptides were identified that act with selectivity on α1β and α3β, compared to α2β, glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol's enhancing actions at the receptor in the absence of zinc. Comparisons of the amino acid sequences of heptapeptides capable of potentiating glycine receptor function revealed several consensus sequences that may be predictive of a peptide's enhancing ability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The glycine receptor (GlyR) is the predominant inhibitory neurotransmitter receptor in the brainstem and spinal cord but is also found in higher brain regions. GlyR function is affected by a variety of allosteric modulators including drugs of abuse, such as ethanol and inhalants and the ubiquitous divalent cation zinc. Two-electrode voltage-clamp experiments were conducted on Xenopus laevis oocytes expressing wild-type α1 homomeric glycine receptors to compare the degree of enhancement produced by zinc on GlyR activated by two agonists (glycine vs. taurine) that vary markedly in their efficacies. Zinc potentiation of both glycine- and taurine-evoked currents was the same at the concentrations of agonists that produced the same currents, corresponding to 6% of the maximal effect of glycine compared to 23% of the maximal effect of taurine. Similar results were seen with 50 and 200 mM ethanol. A direct comparison of agonist concentration-response curves showed that zinc enhancement was greater, overall, for taurine-activated than glycine-activated receptors. In addition, zinc only enhanced taurine- but not glycine-activated GlyR when agonists were applied at saturating concentrations. These data suggest that zinc affects taurine affinity, as well as the probability of channel opening at sub-maximal taurine concentrations, and that the magnitude of allosteric modulation at the GlyR depends on the efficacy of the agonist tested. This has implications for mutagenesis studies in which changes in the degree of allosteric modulation observed may result from mutation-induced changes in agonist efficacy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nM to low μM concentrations, and inhibiting its effects at higher concentrations. Because of zinc's high potency at the glycine receptor, there exists a possibility that effects attributed solely to exogenously-applied glycine in fact contain an undetected contribution of zinc acting as an allosteric modulator. We found that glycine solutions made up in standard buffers and using deionized distilled water produced effects that could be decreased by the zinc chelator tricine. This phenomenon was observed in three different vials tested and persisted even if vials were extensively washed, suggesting the zinc was probably present in the buffer constituents. In addition, polystyrene, but not glass, pipets bore a contaminant that enhanced glycine receptor function and that could also be antagonized by tricine. Our findings suggest that without checking for this effect using a chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Taurine is an endogenous ligand acting on glycine receptors in many brain regions, including the hippocampus, prefrontal cortex, and nucleus accumbens (nAcc). These areas also contain low concentrations of zinc, which is known to potentiate glycine receptor responses. Despite an increasing awareness of the role of the glycine receptor in the rewarding properties of drugs of abuse, the possible interactions of these compounds with zinc has not been thoroughly addressed. Two-electrode voltage-clamp electrophysiological experiments were performed on α1, α2 α1β and α2β glycine receptors expressed in Xenopus laevis oocytes. The effects of zinc alone, and zinc in combination with other positive modulators on the glycine receptor, were investigated when activated by the full agonist glycine versus the partial agonist taurine. Low concentrations of zinc enhanced responses of maximally-effective concentrations of taurine but not glycine. Likewise, chelation of zinc from buffers decreased responses of taurine- but not glycine-mediated currents. Potentiating concentrations of zinc decreased ethanol, isoflurane, and toluene enhancement of maximal taurine currents with no effects on maximal glycine currents. Our findings suggest that the concurrence of high concentrations of taurine and low concentrations of zinc attenuate the effects of additional modulators on the glycine receptor, and that these conditions are more representative of in vivo functioning than effects seen when these modulators are applied in isolation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Taurine acts as a partial agonist at the glycine receptor (GlyR) in some brain regions such as the hippocampus, striatum, and nucleus accumbens. Ethanol, volatile anesthetics, and inhaled drugs of abuse are all known positive allosteric modulators of GlyRs, but their effects on taurine-activated GlyRs remain poorly understood, especially their effects on the high concentrations of taurine likely to be found after synaptic release. Two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes was used to compare the enhancing effects of ethanol, anesthetics, and inhalants on human homomeric α1-GlyR activated by saturating concentrations of glycine versus taurine. Allosteric modulators had negligible effects on glycine-activated GlyR while potentiating taurine-activated currents. In addition, inhaled anesthetics markedly enhanced desensitization rates of taurine- but not glycine-activated receptors. Our findings suggest that ethanol, volatile anesthetics, and inhalants differentially affect the time courses of synaptic events at GlyR, depending on whether the receptor is activated by a full or partial agonist.

Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR α1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I α1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317-329, 2012).

Background: Emerging evidence suggests that taurine acts as a partial agonist on glycine receptors (GlyR) in vitro and in vivo. Ethanol acts as an allosteric modulator on the GlyR producing a leftward shift of the glycine concentration-response curve, with no enhancing effects observed at saturating glycine concentrations. However, to date, no electrophysiological studies have been performed on ethanol modulation of taurine-activated GlyR., Methods: Wild-type alpha1 GlyR, or those bearing a serine-267 to isoleucine replacement (S267I), were homomerically expressed in Xenopus oocytes and voltage clamped at -70 mV. Ethanol was co-applied with varying concentrations of glycine or taurine and the enhancing effects of ethanol compared., Results: Ethanol potentiated glycine- and taurine-activated GlyR responses in a concentration-dependent manner. It shifted taurine and glycine concentration-response curves to the left, having no effects at saturating agonist concentrations. Chelation of zinc by tricine decreased ethanol enhancement of taurine-gated GlyR function. The S267I mutation prevented ethanol enhancement of taurine-mediated responses as previously also reported for glycine., Conclusion: Ethanol modulates taurine activation of GlyR function by a mechanism similar to that of the full agonist glycine. The lack of effect of ethanol at saturating taurine concentrations provides mechanistic information on alcohol actions at the GlyR.

The glycine receptor (GlyR) is a member of the Cys-loop superfamily of ligand-gated ion channels and the major mediator of inhibitory neurotransmission in the spinal cord and brainstem. Many allosteric modulators affect the functioning of members of this superfamily, with some such as benzodiazepines showing great specificity and others such as zinc, alcohols, and volatile anesthetics acting on multiple members. To date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified, hindering both experimental characterization of the receptor and development of GlyR-related therapeutics. We used phage display to identify novel peptides that specifically modulate GlyR function. Peptide D12-116 markedly enhanced GlyR currents at low micromolar concentrations but had no effects on the closely related gamma-aminobutyric acid type A receptors. This approach can readily be adapted for use with other channels that currently lack specific allosteric modulators.

Proper regulation of neurotransmission requires that ligand-activated ion channels remain closed until agonist binds. How channels then open remains poorly understood. Glycine receptor (GlyR) gating is initiated by agonist binding at interfaces between adjacent subunits in the extracellular domain. Aspartate-97, located at the alpha1 GlyR interface, is a conserved residue in the cys-loop receptor superfamily. The mutation of D97 to arginine (D97R) causes spontaneous channel opening, with open and closed dwell times similar to those of maximally activated WT GlyR. Using a model of the N-terminal domain of the alpha1 GlyR, we hypothesized that an arginine-119 residue was forming intersubunit electrostatic bonds with D97. The D97R/R119E charge reversal restored this interaction, stabilizing channels in their closed states. Cysteine substitution shows that this link occurs between adjacent subunits. This intersubunit electrostatic interaction among GlyR subunits thus contributes to the stabilization of the closed channel state, and its disruption represents a critical step in GlyR activation.

Glycine receptor function mediates most inhibitory neurotransmission in the brainstem and spinal cord and is enhanced by alcohols, volatile anesthetics, inhaled drugs of abuse, and endogenous compounds including zinc. Because zinc exists ubiquitously throughout the brain, investigations of its effects on the enhancement of GlyR function by alcohols and anesthetics are important to understanding the effects of these agents in vivo. In the present study, the effects of zinc plus ethanol, pentanol, or isoflurane were tested on homomeric alpha1 glycine receptors to determine if concurrent applications of physiological concentrations of zinc with each of these modulators changed the magnitude of their effects. Homomeric alpha1 glycine receptors were expressed in Xenopus laevis oocytes, and the two-electrode voltage-clamp technique was used to measure glycine-mediated currents in the presence of combinations of zinc with ethanol, pentanol or isoflurane. The combined effects of zinc plus ethanol were greater than the sum of the effects produced by either compound alone. However, this was not seen when zinc was combined with either pentanol or isoflurane. Chelation of zinc by tricine decreased the effects of sub-maximal, but not maximal, concentrations of glycine, and diminished the magnitude of ethanol enhancement observed. These findings suggest a zinc/ethanol interaction at the alpha1 GlyR that results in the enhancement of the effects of ethanol action on GlyR function., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The glycine receptor (GlyR) is a ligand-gated ion channel and member of the nicotinic acetylcholine receptor superfamily. Acting as allosteric modulators of receptor function, drugs such as alcohol and volatile anesthetics enhance the function of GlyRs. The actions of these drugs at inhibitory receptors in the brain and spinal cord are thought to produce many of the physiological effects associated with their use. The actions of ethanol on the GlyR have been well studied on the macroscopic, whole cell level. We examined the effects of 3 microM glycine +/- 50 or 200 mM ethanol on outside-out patches pulled from Xenopus laevis oocytes expressing wild-type alpha1 GlyR, to determine the effects of alcohol at the single-channel level. Alcohol enhanced GlyR function in a very specific manner. It had minimal effects on open and closed dwell times and likelihood. Instead, ethanol potentiated GlyR function almost exclusively by increasing burst durations and increasing the number of channel openings per burst, without affecting the percentage of open time within bursts. Kinetic modeling suggests that ethanol increases burst durations by decreasing the rate of glycine unbinding.

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.

Alcohols and volatile anesthetics modulate the function of cys-loop ligand-gated ion channels, binding to a putative site between transmembrane domains two and three. The extracellular linker between these two domains is important in the transduction of the gating signal from the glycine binding site to the channel gate. Although the anesthetic binding site is proposed to be in the same region throughout the cys-loop receptor family, the modulatory effects of these compounds depend on the receptor. A sequence comparison revealed an extra proline in the TM2-TM3 loop of the 5-HT3A receptor (5-HT3AR) that is not found in the glycine receptor (GlyR). We hypothesized that this proline residue could affect the size and orientation of the putative alcohol and anesthetic binding pocket and perhaps explain some of the differences in alcohol and anesthetic effects seen in this family of receptors. A lysine to proline mutation was introduced into the TM2-TM3 linker region at position 281 (K281P) of the alpha1 GlyR. Mutation at this residue did not affect thiol binding to residues in TM2 or TM3 and it does not appear that residue 281 constitutes part of the alcohol binding site. The K281P receptors displayed constitutive activity in the absence of glycine, and unlike wild-type receptors, this channel opening was antagonized by application of either volatile anesthetics or another GlyR modulator, zinc. Our data suggest that the TM2-TM3 extracellular loop plays a role in the transduction of signals generated by allosteric modulators in addition to gating signals that follow glycine binding.

Alcohols and volatile anesthetics enhance the function of inhibitory glycine receptors (GlyRs). This is hypothesized to occur by their binding to a pocket formed between the transmembrane domains of individual alpha1 GlyR subunits. Because GlyRs are pentameric, it follows that each GlyR contains up to five alcohol/anesthetic binding sites, with one in each subunit. We asked how many subunits per pentamer need be bound by drug in order to enhance receptor-mediated currents. A cysteine mutation was introduced at amino acid serine 267 (S267C) in the transmembrane 2 domain as a tool to block GlyR potentiation by some anesthetic drugs and to provide a means for covalent binding by the small, anesthetic-like thiol reagent propyl methanethiosulfonate. Xenopus laevis oocytes were co-injected with various ratios of wild-type (wt) to S267C alpha1 GlyR cDNAs in order to express heteromeric receptors with a range of wt:mutant subunit stoichiometries. The enhancement of GlyR currents by 200 mm ethanol and 1.5 mm chloroform was positively correlated with the number of wt subunits found in heteromeric receptors. Furthermore, currents from oocytes injected with high ratios of wt to S267C cDNAs (up to 200:1) were significantly and irreversibly enhanced following propyl methanethiosulfonate labeling and washout, demonstrating that drug binding to a single subunit in the receptor pentamer is sufficient to induce enhancement of GlyR currents.

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 alpha1, alpha2 or the mutant alpha1(A52S) GlyRs were measured using two-electrode voltage clamp. Pressure reversibly antagonized potentiation of glycine in alpha1 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 alpha2 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 alpha1(A52S) GlyR findings suggest that increased attention should be focused on the amino terminus as a potential target for ethanol action.

Alcohols and volatile anesthetics affect the function of members of the nicotinic acetylcholine (nACh) superfamily of receptors. Studies on glycine and GABA(A) receptors implicate amino acid residues within transmembrane (TM) regions two and three of these receptors as critical for alcohol and anesthetic enhancement of receptor function. The serotonin-3 (5-HT(3)) receptor is a member of the nicotinic acetylcholine receptor superfamily, sharing sequence and structural homology with the other members. We tested the hypothesis that amino acids of the 5-HT(3) receptor homologous to those shown to affect alcohol and anesthetic potentiation in GABA(A) and glycine receptors also determine the effects of these compounds on the 5-HT(3) receptor. Six 5-HT(3A) mutant cDNAs were generated by site-directed mutagenesis of two amino acids, phenylalanine-269 (14') and lecucine-270 (15') in transmembrane domain two (TM2). When assayed electrophysiologically in Xenopus oocytes, wild-type 5-HT(3) receptors exhibit enhancement of function by enflurane, halothane, isoflurane, chloroform and ethanol, but not by decanol and propofol. Mutations in transmembrane domain two markedly affected alcohol and anesthetic enhancement of 5-HT(3) receptor function. Some mutations had differential effects on the abilities of the isomers enflurane and isoflurane to potentiate 5-HT(3) receptor function.

Strychnine-sensitive glycine receptors (GlyRs) inhibit neurotransmission in the spinal cord and brainstem. To better define the function of this receptor in vivo, we constructed a point mutation that impairs receptor function in the alpha1-subunit and compared these knock-in mice to oscillator (spdot) mice lacking functional GlyR alpha1-subunits. Mutation of the serine residue at amino acid 267 to glutamine (alpha1S267Q) results in a GlyR with normal glycine potency but decreased maximal currents, as shown by electrophysiological recordings using Xenopus oocytes. In addition, single-channel recordings using human embryonic kidney 293 cells indicated profoundly altered properties of the mutated GlyR. We produced knock-in mice bearing the GlyR alpha1 S267Q mutation to assess the in vivo consequences of selectively decreasing GlyR efficacy. Chloride uptake into brain synaptoneurosomes from knock-in mice revealed decreased responses to maximally effective glycine concentrations, although wild-type levels of GlyR expression were observed using 3H-strychnine binding and immunoblotting. A profound increase in the acoustic startle response was observed in knock-in mice as well as a "limb clenching" phenotype. In contrast, no changes in coordination or pain perception were observed using the rotarod or hot-plate tests, and there was no change in GABA(A)-receptor-mediated chloride uptake. Homozygous S267Q knock-in mice, like homozygous spdot mice, exhibited seizures and died within 3 weeks of birth. In heterozygous spdot mice, both decreased 3H-strychnine binding and chloride flux were observed; however, neither enhanced acoustic startle responses nor limb clenching were seen. These data demonstrate that a dominant-negative point mutation in GlyR disrupting normal function can produce a more dramatic phenotype than the corresponding recessive null mutation, and provides a new animal model to evaluate GlyR function in vivo.

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.

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.

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 alpha1 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 alpha1 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.

Glycine receptors (GlyRs) are transmitter-gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR beta subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole-cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the beta subunit was examined using site-directed mutagenesis and expression of recombinant (alpha(1)beta(Y413F)) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine-evoked currents (I(Gly)) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I(Gly) in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2-fold (control EC(50) = 143 microM; cSrc EC(50) = 74 microM). Exogenous cSrc, applied intracellularly, upregulated heteromeric alpha(1)beta receptors but not homomeric alpha(1) receptors. Substitution mutation of the tyrosine to phenylalanine at position beta-413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down-regulated wild-type alpha(1)beta but not alpha(1)beta(Y413F) receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine-413 residue of the beta subunit.

Unlabelled: The in vivo potencies of anesthetics correlate with their capacity to suppress the reaction of luciferin with luciferase. In addition, luciferin has structural resemblances to etomidate. These observations raise the issues of whether luciferin, itself, might affect anesthetic requirement, and whether luciferase resembles the site of anesthetic action. Because the polar luciferin is unlikely to cross the blood-brain barrier (we found that the olive oil/water partition coefficient was 100 +/- 36 x 10(-7)), we studied these issues in rats by measuring the effect of infusion of luciferin in artificial cerebrospinal fluid into the lumbar subarachnoidal space and into the cerebral intraventricular space on the MAC (the minimum alveolar anesthetic concentration required to eliminate movement in response to a noxious stimulus in 50% of tested subjects) of isoflurane. MAC in rats given lumbar intrathecal doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate, did not differ significantly from MAC in rats receiving only artificial cerebrospinal fluid into the lumbar intrathecal space. MAC slightly decreased when doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate were infused intraventricularly (P < 0.05). In contrast to the absent or minimal effects of luciferin, intrathecal or intraventricular infusion of etomidate at similar or smaller doses significantly decreased isoflurane MAC. Luciferin did not affect +-aminobutyric acid type A or acetylcholine receptors expressed in Xenopus oocytes. These results suggest that luciferin has minimal or no anesthetic effects. It also suggests that luciferin/luciferase may not provide a good surrogate for the site at which anesthetics act, if this site is on the surface of neuronal cells., Implications: In proportion to their potencies, anesthetics inhibit luciferin's action on luciferase, and luciferin structurally resembles the anesthetic etomidate. However, in contrast to etomidate, luciferin given intrathecally or into the third cerebral ventricle does not have anesthetic actions, and it does not affect +-aminobutyric acid or acetylcholine receptors in vitro. Luciferase may not provide a good surrogate for the site at which anesthetics act.

We have carried out a detailed sequence and functional analysis of a novel human facilitative glucose transporter, designated GLUT10, located in the Type 2 diabetes-linked region of human chromosome 20q12-13.1. The GLUT10 gene is located between D20S888 and D20S891 and is encoded by 5 exons spanning 26.8 kb of genomic DNA. The human GLUT10 cDNA encodes a 541 amino acid protein that shares between 31 and 35% amino acid identity with human GLUT1-8. The predicted amino acid sequence of GLUT10 is nearly identical in length to the recently described GLUT9 homologue, but is longer than other known members of the GLUT family. In addition, we have cloned the mouse cDNA homolog of GLUT10 that encodes a 537 amino acid protein that shares 77.3% identity with human GLUT10. The amino acid sequence probably has 12 predicted transmembrane domains and shares characteristics of other mammalian glucose transporters. Human and mouse GLUT10 retain several sequence motifs characteristic of mammalian glucose transporters including VP497ETKG in the cytoplasmic C-terminus, G73R[K,R] between TMD2 and TMD3 (PROSITE PS00216), VD92RAGRR between TMD8 and TMD9 (PROSITE PS00216), Q242QLTG in TMD7, and tryptophan residues W430 (TMD10) and W454 (TMD11), that correspond to trytophan residues previously implicated in GLUT1 cytochalasin B binding and hexose transport. Neither human nor mouse GLUT10 retains the full P[E,D,N]SPR motif after Loop6 but instead is replaced with P186AG[T,A]. A PROSITE search also shows that GLUT10 has lost the SUGAR TRANSPORT 2 pattern (PS00217), a result of the substitution G113S in TMD4, while all other known human GLUTs retain the glycine and the pattern match. The significance of this substitution is unknown. Sites for N-linked glycosylation are predicted at N334ATG between TMD8 and TMD9 and N526STG in the cytoplasmic C-terminus. Northern hybridization analysis identified a single 4.4-kb transcript for GLUT10 in human heart, lung, brain, liver, skeletal muscle, pancreas, placenta, and kidney. By RT-PCR analysis, GLUT10 mRNA was also detected in fetal brain and liver. When expressed in Xenopus oocytes, human GLUT10 exhibited 2-deoxy-D-glucose transport with an apparent Km of approximately 0.3 mM. D-Glucose and D-galactose competed with 2-deoxy-D-glucose and transport was inhibited by phloretin. The gene localization and functional properties suggest a role for GLUT10 in glucose metabolism and Type 2 diabetes., (Copyright 2001 Academic Press.)

Recent studies suggest that alcohols, volatile anesthetics, and inhaled drugs of abuse, which enhance gamma-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 alpha(1) 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.

1. Each residue in the second transmembrane segment (TM2) of the human GABA(A) receptor alpha(2) subunit was individually mutated to tryptophan. The wild-type or mutant alpha(2) subunits were expressed with the wild-type human GABA(A) receptor beta(2) subunit in Xenopus oocytes, and the effects of these mutations were investigated using two-electrode voltage-clamp recording. 2. 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 alpha(2)(V257W)beta(2) mutant receptor, which was not sensitive to picrotoxin. 3. 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 alpha(2)beta(2) 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. 4. 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. 5. These results are consistent with a unique role for S270 in the action of ethanol within the TM2 region, and with models of GABA(A) 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.

Inhalable solvents possess significant abuse liability and produce many of the neurobehavioral effects typically associated with central nervous system-depressant agents, including motor incoordination, anxiolysis, and the elicitation of signs of physical dependence on withdrawal. We tested the hypothesis that the commonly abused solvents toluene, 1,1,1-trichloroethane (TCE), and trichloroethylene (TCY) affect ligand-gated ion channel activity, as do other classes of central nervous system-depressive agents. TCE and toluene, like ethanol, reversibly enhanced gamma-aminobutyric acid (GABA)(A) receptor-mediated synaptic currents in rat hippocampal slices. All three inhalants significantly and reversibly enhanced neurotransmitter-activated currents at alpha1beta1 GABA(A) and alpha1 glycine receptors expressed in Xenopus oocytes. We previously identified specific amino acids of glycine and GABA(A) receptor subunits mediating alcohol and volatile anesthetic enhancement of receptor function. Toluene, TCE, and TCY were tested on several glycine receptor mutants, some of which were insensitive to ethanol and/or enflurane. Toluene and TCY enhancement of glycine receptor function was seen in all these mutants. However, the potentiating effects of TCE were abolished in three mutants and enhanced in two, a pattern more akin to that seen with enflurane than ethanol. These data suggest that inhaled drugs of abuse affect ligand-gated ion channels, and that the molecular sites of action of these compounds may overlap with those of ethanol and the volatile anesthetics.

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 alpha1 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.

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a "cutoff" is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the alpha subunit. We now demonstrate that these residues in the glycine alpha1 and the gamma-aminobutyric acid rho1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine alpha1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the gamma-aminobutyric acid rho1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

Glycine and gamma-aminobutyric acid (GABA)A receptors are members of the "superfamily" of ion channels, and are sensitive to allosteric modulation by n-alcohols such as ethanol and butanol. We recently demonstrated that the mutation of Ser-267 to Ile in the alpha1 subunit abolished ethanol regulation of glycine receptors (Gly-R). In the present study, a pair of chimeric receptors was studied, in which a 45-amino acid domain comprising transmembrane domains 2 and 3 was exchanged between the Gly-Ralpha1 and gamma-aminobutyric acid rho1 subunits. Detailed pharmacologic analysis of these chimeras confirmed that this domain of the Gly-R confers enhancement of receptor function by ethanol and butanol. An extensive series of mutations at Ser-267 in the Gly-Ralpha1 subunit was also prepared, and the resulting homomeric receptors were expressed and tested for sensitivity to glycine, and allosteric modulation by alcohols. All of the mutant receptors expressed successfully in Xenopus oocytes. Mutation of Ser-267 to small amino acid residues such as Gly or Ala produced receptors in which glycine responses were potentiated by ethanol. As we have reported previously, the mutant Gly-Ralpha1 (Ser-267 --> Ile) was completely insensitive to ethanol; mutation of Ser-267 to Val had a similar effect. Mutation of Ser-267 to large residues such as His, Cys, or Tyr resulted in inhibition of Gly-R function by ethanol. These results demonstrate that the size of the amino acid residue at position alpha267 plays a crucial role in determining the functional consequences of allosteric modulation of the Gly-R by alcohols.

Cytoclean a commercially available detergent, has selective actions on ligand-gated ion channels. Cytoclean (0.0005-0.01% v/v) potentiated 50 microM glycine responses in oocytes expressing alpha-2 glycine receptors by 23 +/- 7% to 342 +/- 43%. Cytoclean is composed of five components dissolved in water, but only one reagent, Bio-Soft D-62, modulated responses of oocytes expressing alpha-2 glycine receptors. Bio-Soft D-62 (0.00005-0.001% w/v), potentiated 50 microM glycine responses by 13 +/- 1% to 474 +/- 50%. Bio-Soft D-62 is composed of linear alkylbenzene sulfonate (> 95% C12 chain). The effects of Cytoclean or Bio-Soft D-62 were examined on alpha-1 beta-2 and alpha-1 beta-2 gamma-2L gamma-aminobutyric acidA, gamma-aminobutyric acid rho 1, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid, kainate and 5-hydroxytryptamine3 receptors expressed in Xenopus laevis oocytes. Enhancement of gamma-aminobutyric acidA receptor function ranged from approximately 21% to 458% with Cytoclean (0.0001-0.01%), respectively. Bio-Soft D-62 (0.001%) inhibited GABA rho 1 receptor function by approximately 72%. Cytoclean had no effect on 5-hydroxytryptamine3 or GluR6 function, but Cytoclean (0.005% and 0.01%) inhibited GluR3-mediated currents by approximately 21% and approximately 41%, respectively. These results suggest that trace amounts of Cytoclean, such as amounts adhering to glassware, may modulate ion channel function and potentially confound experimental results.

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 gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 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 GABA(A) 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.

GABAA receptors composed of human alpha 1 beta 2 gamma 2L, alpha 1 beta 2 gamma 2S, alpha 1 beta 3 gamma 2S, alpha 6 beta 3 gamma 2S, and alpha 5 beta 3 gamma 3 subunits as well as bovine alpha 1 beta 1 gamma 2L and alpha 1 beta 1 subunits were stably expressed in mammalian L(tk-) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a 36Cl- flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk-) cells than oocytes; this difference ranged from 2.6-to 26-fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk-) cells, low (5-50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the gamma 2L subunit. In oocytes, concentrations of 30-100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except alpha 6 beta 3 gamma 2S and alpha 1 beta 1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk-) cells and oocytes, except for alpha 6 beta 3 gamma 2S where the pentobarbital enhancement was much greater in oocytes than cells. The alpha 6 beta 3 gamma 2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABAA receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.

The neurotransmitter gamma-aminobutyric acid (GABA) inhibits the activity of signal-receiving neurons by interacting with the GABAA receptor on these cells. The GABAA receptor is a channel-forming protein that allows the passage of chloride ions into the cells. Excessive GABAA activation may play a role in mediating the sedative effects of alcohol and other sedating and anesthetic agents. For example, alcohol enhances the GABAA-mediated chloride flow into cells and may thereby enhance neuronal inhibition. Alcohol's effects on the GABAA-receptor function likely involve other molecules (e.g., other neurotransmitters and proteins that add phosphate groups to the receptor [i.e., protein kinases]). Several experimental approaches also have suggested that changes in GABAA-receptor function contribute to the tolerance to and dependence on alcohol. Finally, individual differences in the GABA system may play a role in determining a person's susceptibility to developing alcohol dependence.

Effects of ethanol on strychnine-sensitive glycine receptors were studied in Xenopus laevis oocytes expressing alpha 1 wild-type, alpha 2, or mutant alpha 1(A52S) homomeric glycine receptors. This alpha 1(A52S) mutant, in which a serine residue substitutes for alanine at amino acid 52, is responsible for the spasmodic phenotype in mice and alters the ability of glycine to activate the receptor. Pharmacologically relevant concentrations of ethanol (10-200 mM) reversibly potentiated the glycine receptor function in all receptors. Ethanol potentiation depended on the glycine concentration used, with decreased potentiation observed at higher glycine concentrations. Homomeric alpha 1 glycine receptors were more sensitive to the effects of ethanol than were alpha 2 or the mutant alpha 1(A52S) receptors. No differences were found in ethanol sensitivity between alpha 2 and the mutant alpha 1(A52S) receptors. The alpha 2 subunit has a threonine residue, a conservative substitution for serine, at amino acid 52. The general anesthetic propofol was also tested in homomeric alpha 1, alpha 2, or the mutant alpha 1(A52S) receptors. Propofol, at unaesthetic concentrations (1-5 microM), reversibly potentiated the glycine receptor function in a concentration-dependent manner and to an equal extent in the three subunits tested. These data suggest that the mutation of an alanine to serine at amino acid 52 of the alpha subunit is responsible for the difference in ethanol sensitivity seen in homomeric receptors composed of alpha 1 and alpha 2 subunits.

1. The effects of n-alcohols on GABAA and glutamate receptor systems were examined, and in vitro effectiveness was compared with in vivo effects in mice and tadpoles. We expressed GABAA, NMDA, AMPA, or kainate receptors in Xenopus oocytes and examined the actions of n-alcohols on receptor function using two-electrode voltage clamp recording. 2. The function of GABAA receptors composed of alpha 1 beta 1 or alpha 1 beta 1 gamma 2L subunits was potentiated by all of the n-alcohols studied (butanol-dodecanol). 3. In contrast to GABAA receptors, glutamate receptors expressed from mouse cortical mRNA or from cRNAs encoding AMPA (GluR3)- or kainate (GluR6)-selective subunits were much less sensitive to longer chain alcohols. In general, octanol and decanol were either without effect or high concentrations were required to produce inhibition. 4. In contrast to the lack of behavioural effects by long chain alcohols reported previously, decanol produced loss of righting reflex in short- and long-sleep mice, indicating that the in vivo effects of decanol may be due in part to actions at GABAA receptors. Furthermore, butanol, hexanol, octanol, and decanol produce similar potentiation of GABAA receptor function at concentrations required to cause loss of righting reflex in tadpoles, an in vivo model where alcohol distribution is not a compromising factor. 5. Thus, the in vivo effects of long chain alcohols are not likely to be due to their actions on NMDA, AMPA, or kainate receptors, but may be due instead to potentiation of GABAA receptor function.