Transgenic expression of a mutant glycine receptor decreases alcohol sensitivity of mice.

Glycine receptors (GlyRs) are pentameric ligand-gated ion channels that inhibit neurotransmission in the adult brainstem and spinal cord. GlyR function is potentiated by ethanol in vitro, and a mutant GlyR subunit alpha(1)(S267Q) is insensitive to the potentiating effects of ethanol. To test the importance of GlyR for the actions of ethanol in vivo, we constructed transgenic mice with this mutation. Under the control of synapsin I regulatory sequences, transgenic expression of S267Q mutant GlyR alpha(1) subunits in the nervous system was demonstrated using [(3)H]strychnine binding and immunoblotting. These mice showed decreased sensitivity to ethanol in three behavioral tests: ethanol inhibition of strychnine seizures, motor incoordination (rotarod), and loss of righting reflex. There was no change in ethanol sensitivity in tests of acute functional tolerance or body temperature, and there was no change in ethanol metabolism. Transgene effects were pharmacologically specific for ethanol, compared with pentobarbital, flurazepam, and ketamine. These results support the idea that glycine receptors contribute to some behavioral actions of ethanol and that ethanol sensitivity can be changed in vivo by transgenic expression of a single receptor subunit.

Does acetaldehyde mediate ethanol action in the central nervous system?

BACKGROUND: Some of the effects of ethanol in the central nervous system are due to changes in function of ligand-gated ion channels. Production of detectable amounts of acetaldehyde, a primary metabolite of ethanol, has been demonstrated in brain homogenates. The aim of this study was to determine whether central actions that are often attributed to ethanol may actually be mediated by acetaldehyde. METHODS: The effects of acetaldehyde (1-1000 microM) were tested by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing 10 different ligand-gated ion channel receptors [alpha1 glycine; alpha1beta2gamma2Sgamma-aminobutyric acid (GABA)A; rho1 GABAc; 5-hydroxytryptamine-3A; NR1a/NR2A NMDA; GluR1/GluR2 AMPA; GluR6/KA2 kainate; and alpha4beta2, alpha4beta4, and alpha2beta4 nicotinic-acetylcholine] and the G-protein-coupled inward rectifying potassium channel GIRK2. We also investigated the effect of acetaldehyde on the dopamine transporter (DAT), performing dopamine uptake assays in oocytes expressing DAT. RESULTS: Acetaldehyde (1 and 10 microM) significantly enhanced alpha1 glycine receptor-mediated currents. Acetaldehyde did not affect the function of any of the other receptors tested or the potassium currents measured in GIRK2 channels. Moreover, acetaldehyde did not alter the DAT-mediated dopamine uptake. CONCLUSIONS: Our results suggest a potential minor role for acetaldehyde in the glycine receptor-mediated effects of ethanol. Otherwise, acetaldehyde does not modulate function of the neuronal receptors tested in this study, in GIRK channels or DAT, when expressed recombinantly in Xenopus laevis oocytes.

Alcohol actions on GABA(A) receptors: from protein structure to mouse behavior.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were R. Adron Harris and Susumu Ueno. The presentations were (1) Protein kinase Cepsilon-regulated sensitivity of gamma-aminobutyric acid type A (GABAA) receptors to allosteric agonists, by Robert O. Messing, A. M. Sanchez-Perez, C. W. Hodge, T. McMahon, D. Wang, K. K. Mehmert, S. P. Kelley, A. Haywood, and M. F. Olive; (2) Genetic and functional analysis of a GABAA receptor gamma2 subunit variant: A candidate for quantitative trait loci involved in alcohol sensitivity and withdrawal, by Kari J. Buck and Heather M. Hood; (3) Tryptophan-scanning mutagenesis in GABAA receptor subunits: Channel gating and alcohol actions, by Susumu Ueno; and (4) Can a single binding site account for actions of alcohols on GABAA and glycine receptors? by R. Adron Harris, Yuri Blednov, Geoffrey Findlay, and Maria Paola Mascia.

The anesthetic potency of propanol and butanol versus propanethiol and butanethiol in alpha1 wild type and alpha1(S267Q) glycine receptors.

UNLABELLED: Although similar in shape and size, and although differing only by substitution of a sulfur atom for an oxygen atom, propanethiol and butanethiol differ markedly from propanol and butanol in their in vivo potency and physical properties. Recent theories of narcosis suggest that anesthetics may act by enhancing the effect of inhibitory agonists, such as glycine, on their receptors. We tested whether propanol, butanol, propanethiol, and butanethiol enhance the effect of glycine on alpha1 glycine receptors expressed in Xenopus laevis oocytes in a manner that reflects the in vivo differences found for potencies. As anticipated, we found an immediate parallel between in vivo (rat minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects) and in vitro (recombinant receptor) effects. All four compounds enhanced the effect of glycine on wild type receptors, and the extent of enhancement for a given minimum alveolar concentration-multiple was approximately the same for all compounds. We also found that propanethiol, butanethiol, propanol, and butanol did not affect, or minimally affected, the action of glycine in anesthetic resistant mutants in which the amino acid serine at position 267 was replaced by glutamine [alpha1(S267Q)]. IMPLICATIONS: The in vivo potencies of propanethiol, butanethiol, propanol, and butanol correlate with their capacities to enhance the effect of glycine on alpha1 glycine receptors expressed in Xenopus laevis oocytes. These results support the notion that a protein mediates anesthetic action.

The anesthetic potencies of alkanethiols for rats: relevance to theories of narcosis.

UNLABELLED: Meyer and Overton suggested that anesthetic potency correlates inversely with lipophilicity. Thus, MAC times the olive oil/gas partition coefficient equals an approximately constant value of 1.82 +/- 0.56 atm (mean +/- SD). MAC is the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects. Although MAC times the olive oil/gas partition coefficient also equals an approximately constant value for normal alkanols from methanol through octanol, the value (0.156 +/- 0.072 atm) is 1/10th that found for conventional anesthetics. We hypothesized that substitution of sulfur for the oxygen in n-alkanols would decrease their saline/gas partition coefficients (i.e., decrease polarity) while sustaining lipid/gas partition coefficients. Further, we hypothesized that these changes would produce products of MAC times olive oil partition coefficients that approximate those of conventional anesthetics. To test these predictions, we measured MAC in rats, and saline and olive oil solubilities for the series H(CH(2))(n)SH, comparing the results with the series H(CH(2))(n)OH for compounds having three to six carbon atoms. As hypothesized, the alkanethiols had similar oil/gas partition coefficients, 1000-fold smaller saline gas partition coefficients, and MAC values 30 times greater than for comparable alkanols. Such findings are consistent with the notion that the greater potency of many alkanols (greater than would be predicted from conventional inhaled anesthetics and the Meyer-Overton hypothesis) results from their greater polarity. IMPLICATIONS: The in vivo anesthetic potency of alkanols and alkanethiols correlates with their lipophilicity and hydrophilicity.

Behavioural changes produced by transgenic overexpression of gamma2L and gamma2S subunits of the GABAA receptor.

Transgenic mice overexpressing either the mouse gamma2L or gamma2S subunit of the GABAA receptor were generated in a C57BL/6 J x DBA/2 J mixed background and expanded into transgenic lines. Transgenic mice and littermate controls were analysed with respect to altered behaviour indicative of anxiety, motor activity and acute effects of benzodiazepines and alcohol, as well as with regard to altered responses to alcohol withdrawal and acute functional tolerance to alcohol. Biochemical tests assessed flunitrazepam- and ethanol-enhanced 36Cl- flux stimulated by muscimol in cerebellar and cortical microsacs and [3H]-flunitrazepam binding to cerebellar membranes. There were no significant differences in any of these measures between the transgenic and control mice, except in tests of acute functional tolerance to acute injection of ethanol. Compared to controls, mice carrying either the gamma2L or gamma2S transgene developed significantly less tolerance to the ataxic effects of ethanol. We conclude that acute functional tolerance to ethanol is very sensitive to the amount of GABAA receptor gamma2 subunit available (regardless of whether it is gamma2L or gamma2S) but overexpression of neither subunit isoform alters other behavioural and biochemical phenotypes.

Specific binding sites for alcohols and anesthetics on ligand-gated ion channels.

Ligand-gated ion channels are a target for inhaled anesthetics and alcohols in the central nervous system. The inhibitory strychnine-sensitive glycine and gamma-aminobutyric acid type A receptors are positively modulated by anesthetics and alcohols, and site-directed mutagenesis techniques have identified amino acid residues important for the action of volatile anesthetics and alcohols in these receptors. A key question is whether these amino acids are part of an alcohol/anesthetic-binding site. In the present study, we used an alkanethiol anesthetic to covalently label its binding site by mutating selected amino acids to cysteine. We demonstrated that the anesthetic propanethiol, or alternatively, propyl methanethiosulfonate, covalently binds to cysteine residues introduced into a specific second transmembrane site in glycine receptor and gamma-aminobutyric acid type A receptor subunits and irreversibly enhances receptor function. Moreover, upon permanent occupation of the site by propyl disulfide, the usual ability of octanol, enflurane, and isoflurane to potentiate the function of the ion channels was lost. This approach provides strong evidence that the actions of anesthetics in these receptors are due to binding at a single site.

Enhancement of glycine receptor function by ethanol: role of phosphorylation.

1. The effects of several kinase inhibitors (staurosporine, GF 109203X, H89, KN62, genistein) and of the phosphatase inhibitor calyculin A were studied on the ethanol potentiation and on the function of homomeric alpha1 glycine receptor expressed in Xenopus oocytes using a two electrode voltage clamp recording technique. 2. The function of the homomeric alpha1 glycine receptor was not modified in Xenopus oocytes pretreated with kinase inhibitors or with the phosphatase inhibitor calyculin A. 3. The potentiation of the glycine receptor function induced by ethanol (10-200 mM) was significantly reduced in Xenopus oocytes pretreated with the PKC inhibitors staurosporine or GF 109203X. 4. No differences in propofol (2.5 microM) or halothane (250 microM) actions were found after exposure of Xenopus oocytes to staurosporine. 5. No differences in ethanol sensitivity were found after exposure of Xenopus oocytes expressing glycine alpha1 receptors to H89, KN62, genistein or to the phosphatase inhibitor calyculin A. 6. The mutant alpha1 (S391A), in which the PKC phosphorylation site at serine 391 was mutated to alanine, was less sensitive to the effects of ethanol than was the alpha1 wild type receptor. Moreover, the ethanol potentiation of the glycine receptor function was not affected by treatment with staurosporine in oocytes expressing alpha1 (S391A). 7. The splice variant of the alpha1 glycine receptor subunit, alpha1ins, containing eight additional amino acids and a potential phosphorylation site for PKA, did not differ from wild type for sensitivity to ethanol. 8. These results indicate that phosphorylation by PKC of the homomeric alpha1 glycine receptor subunit modulates ethanol potentiation, but not the function of the glycine receptor.

Mutations of gamma-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor?

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.

Enhancement of glycine receptor function by ethanol is inversely correlated with molecular volume at position alpha267.

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.

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 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.

Glycine receptors from long-sleep and short-sleep mice: genetic differences in drug sensitivity.

The long-sleep (LS) and short-sleep (SS) mice were selected for differences in sensitivity to ethanol but also differ in response to propofol and some neurosteroids. To determine the role of strychnine-sensitive glycine receptors in genetic differences between these mice, effects of propofol, ethanol and pregnenolone sulfate on glycine responses were compared in Xenopus oocytes expressing mRNA extracted from spinal cord of LS and SS mice. The two lines of mice did not differ in sensitivity to glycine, ethanol or pregnenolone sulfate. However, receptors expressed from LS mRNA were more sensitive to the potentiation induced by propofol than those from SS. Binding of [3H]strychnine to spinal cord membranes demonstrated a similar affinity and density of receptors in LS and SS. These results suggest that glycine receptor function could account for differences in propofol sensitivity between LS and SS mice, but may not be responsible for the differences in behavioral sensitivity to ethanol or steroids previously reported.

Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics.

1. The effects of n-alcohols (ethanol to dodecanol) and anaesthetics on strychnine-sensitive glycine receptors were studied in Xenopus oocytes expressing homomeric alpha 1 or alpha 2 glycine receptor subunits, with the two electrode voltage-clamp recording technique. 2. The glycine-induced chloride conductance of homomeric alpha glycine receptors was potentiated by all the alcohols tested when an EC2 concentration of glycine was used. Homomeric alpha 1 and alpha 2 receptors were potentiated similarly by the n-alcohols, except that low concentrations of ethanol produced greater potentiation with alpha 1, as previously reported. 3. Increasing the n-alcohol carbon number has been shown to increase the potency of the alcohols up to decanol at concentrations corresponding to EC50s for producing loss of righting reflex in tadpoles. However, dodecanol was no more potent than decanol, and only modest potentiation (30-60%) was obtained with dodecanol, in contrast to marked (150-200%) potentiation with the other alcohols. Thus, a "cut-off' occurred at about dodecanol. 4. Propofol, alphaxalone, pentobarbitone, halothane and enflurane, reversibly potentiated the function of homomeric alpha 1 glycine receptors at concentrations which represent approximately twice the EC50 for production of anaesthesia in mammals, but ketamine and etomidate were ineffective. 5. Two novel cyclobutane compounds were tested; the anaesthetic compound (1-chloro-1,2,2-trifluorocyclobutane) from 0.5 to 5 mM potentiated the action of glycine in a concentration-dependent manner; however, the non-anaesthetic analogue (1,2-dichloro-hexfluorocyclobutane) had no effect on glycine receptor function at concentrations (25 to 80 microM) predicted to be anaesthetic, based on the lipid solubility of this compound. 6. These results suggest that the alpha subunits of strychnine-sensitive glycine receptors contain sites of action for n-alcohols, propofol, alphaxalone, pentobarbitone and volatile anaesthetics, but not for ketamine and etomidate. Potentiation of glycine receptor function may contribute to the anaesthetic action of n-alcohols and volatile agents.

A single amino acid determines differences in ethanol actions on strychnine-sensitive glycine receptors.

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.

Chronic ethanol intoxication enhances [3H]CCPA binding and does not reduce A1 adenosine receptor function in rat cerebellum.

The effects of acute and chronic treatment with ethanol on the function of A1 adenosine receptor in the rat cerebellar cortex were investigated. Acute administration of ethanol (0.5-5 g/kg) had no effect on the binding of the A1-receptor agonist [3H]2-chloro-N6-cyclopentyladenosine ([3H]CCPA) or that the antagonist [3H]8-cyclopentyl-1-3-dipropylxanthine ([3H]DPCPX) in rat cerebellar cortical membranes. Rats were rendered ethanol dependent by repeated forced oral administration of ethanol (12-18 g/kg per day) for 6 days. [3H]CCPA binding was increased by 23% in cerebellar cortical membranes prepared from rats killed 3 h after ethanol withdrawal compared with saline-treated animals. The increase in [3H]CCPA binding was still apparent 12-24 h after the last ethanol administration, but was no longer detectable 3-6 days after ethanol withdrawal. In contrast, the binding of [3H]DPCPX was not modified in the cerebellar cortex of rats killed at various times after ethanol withdrawal. The acute administration of CCPA [0.25-1 mg/kg, intraperitoneally (IP)] suppressed the tremors and audiogenic seizures apparent 24 h after ethanol withdrawal. Moreover, repeated coadministration of CCPA (0.5 mg/kg, IP, four times daily) and ethanol did not prevent the generation of audiogenic seizures during withdrawal but completely prevented mortality. Finally, CCPA antagonized with similar potencies and efficacies the isoniazid-induced convulsions observed in control and ethanol-withdrawn rats. These results indicate that long-term treatment with intoxicating doses of ethanol enhances [3H]CCPA binding but does not reduce the anticonvulsant efficacy of CCPA or the function of A1 adenosine receptors.

GABAA receptor function and binding in stably transfected cells: chronic ethanol treatment.

Effects of chronic ethanol exposure on GABAA receptors may contribute to tolerance and dependence to alcohol. Ethanol treatment of mice and rats can produce alterations of GABAA receptor binding, function, and subunit mRNA and protein levels. We treated a cell line (PA3 cells) that stably expresses GABAA receptors chronically with ethanol. Expression of bovine alpha 1, beta 1, and gamma 2L GABAA receptor subunits genes in these cells is controlled by a dexamethasone-sensitive promoter, and this provides an excellent system to study the regulation by chronic ethanol treatment of receptors with a defined subunit composition. The actions of the GABA agonist muscimol on receptor function (36Cl- uptake) were not affected by 100 mM ethanol treatment for 4 days, but the actions of flunitrazepam (1 microM) were decreased in cells treated with ethanol. The functional coupling between benzodiazepine and GABA sites on the receptors was affected by chronic ethanol treatment in a manner consistent with results from mice. Ethanol treatments (50 or 100 mM) for 4 days did not affect the affinity (Kd) or receptor density (Bmax) of [3H]flunitrazepam binding, or the levels of alpha 1 subunit mRNA, or alpha 1 or beta 1 subunit proteins. These results demonstrate that the regulation of the stably expressed GABAA receptors by chronic ethanol, in the absence of neuronal receptor gene promoters, is posttranscriptional and likely posttranslational.

Regulation of allosteric coupling and function of stably expressed gamma-aminobutyric acid (GABA)A receptors by chronic treatment with GABAA and benzodiazepine agonists.

Chronic treatments with drugs that stimulate or potentiate gamma-aminobutyric acid (GABA)A receptor function cause uncoupling of allosteric sites and downregulation of the GABAA receptors expressed in neurons. To study these effects on receptors having a defined subunit composition, we treated stably transfected mouse Ltk- cells (PA3 cells) with drugs known to uncouple GABAA receptors. Because dexamethasone controls the expression of bovine alpha-1, beta-1 and gamma 2L GABAA receptor subunit genes in PA3 cells, this expression system provides a way to study receptors in the absence of neuronal subunit gene promoters. We assayed binding of [3H]flunitrazepam to measure allosteric coupling and uptake of 36Cl- to measure GABAA receptor function. Chronic (4 day) treatment of PA3 cells with muscimol, GABA or flunitrazepam reduced the GABA enhancement of binding of [3H]flunitrazepam to PA3 cells. Chronic flurazepam or muscimol treatments also caused downregulation of benzodiazepine potentiation of muscimol-stimulated 36Cl- uptake. Chronic treatment with muscimol did not affect levels of subunit mRNAS and alpha 1- or beta 1-subunit protein of GABAA receptors and chronic flunitrazepam did not affect subunit mRNAs or alpha 1 protein. We conclude that chronic drug treatments regulate allosteric coupling and function of GABAA receptors in stably transfected cells and this regulation cannot be attributed to decreases in the expression of receptor subunits or to expression of subunits other than alpha 1 beta 1 or gamma 2L.

Actions of the general anesthetic propofol on recombinant human GABAA receptors: influence of receptor subunits.

The intravenous general anesthetic 2,6-diisopropylphenol (propofol) potentiates GABAA receptor function, but the precise mechanisms and specificity of this action are still unclear. To study the influence of receptor subunit composition on the action of propofol, 18 different combinations of cloned cDNAs coding for human brain subunit isoforms of the GABAA receptor, as well as mRNAs from mouse brain, were expressed in Xenopus oocytes, and effects of this anesthetic were investigated by the voltage-clamp technique. We found that low concentrations (1-10 microM) of propofol dramatically potentiated GABA-evoked Cl- currents in all GABAA receptor constructs tested. This action did not require specific subunits but was correlated inversely with the GABA sensitivity of each receptor construct. Larger concentrations (10-25 microM) of propofol produced direct activation of Cl- currents, and this action was dependent on the expression of the beta-subunit of the GABAA receptor and did not correlate with the GABA sensitivity of the receptors. These results suggest that propofol exerts a dual effect on GABAA receptors: a positive modulation of the GABA-mediated action on GABAA receptors that is not influenced by the receptor subunit composition, and a specific interaction with the beta-subunit that directly activates the GABAA receptor-coupled Cl- channel.

Ethanol increases GABAA responses in cells stably transfected with receptor subunits.

Ethanol enhancement of GABAA receptor function has been found in some, but not all, studies. These results suggest the existence of ethanol-sensitive and -resistant receptors that may differ in subunit composition, although methodological differences (e.g., 36Cl- flux versus membrane currents) could also contribute to the different results. To examine these possibilities, we used mouse L(tk-) cells stably transfected with alpha 1 + beta 1 or alpha 1 + beta 1 + gamma 2L GABAA receptor subunit DNAs and compared 36Cl- flux with whole-cell, patch-clamp measurements of GABAA receptor function. Both techniques detected a similar modulation of the GABA receptor by ethanol, flunitrazepam, and pentobarbital. The potentiating action of ethanol required the gamma-subunit and was maximal at a concentration of 10 mM. Similar ethanol potentiation was obtained with brief (20 msec) or long (2 sec) applications of GABA. Analysis of data obtained from individual cells expressing alpha 1 beta 1-gamma 2L subunits showed considerable variability in sensitivity to ethanol, particularly with concentrations of 30 and 100 mM. Ethanol potentiated GABA action if the cells were grown on coverslips coated with polylysine, but had no effect on GABAA receptors of cells grown on uncoated coverslips. Thus, ethanol action was influenced by the growth matrix. Taken together, these data indicate that a gamma-subunit is necessary, but not sufficient, for ethanol sensitivity in this cell system. We suggest that posttranslational processing, particularly receptor phosphorylation, may also be important and that stably transfected cells will be useful in elucidating these events.

2-Chloro-N6-cyclopentyladenosine (CCPA), an adenosine A1 receptor agonist, suppresses ethanol withdrawal syndrome in rats.

The ability of 2-chloro-N6-cyclopentyladenosine (CCPA) to suppress ethanol withdrawal syndrome was tested in male rats rendered physically dependent on ethanol by intragastric administrations of ethanol (12-18 g/kg daily for 6 days). CCPA administered 24 hr after the last ethanol dose produced a dose-dependent inhibition of withdrawal signs such as tremors and audiogenically induced seizures, an effect prevented by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX).