A potential cost of evolving epibatidine resistance in poison frogs.

BACKGROUND: Some dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor ß2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4ß2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration-response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the ß2 S108C into the α4ß2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs. RESULTS: The introduction of S108C into the α4ß2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4ß2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity. CONCLUSIONS: The most parsimonious explanation of our current and previous work is that the S108C substitution renders the ß2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while ß2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4ß2 receptor function.

A potential cost of evolving epibatidine resistance in poison frogs.

Background: Some poison arrow frogs sequester the toxin epibatidine as a defense against predators. We previously identified a single amino acid substitution (S108C) at a highly conserved site in a neuronal nicotinic acetylcholine receptor (nAChR) ß2 subunit that prevents epibatidine from binding to this receptor. When placed in a homologous mammalian nAChR this substitution minimized epibatidine binding but also perturbed acetylcholine binding, a clear cost. However, in the nAChRs of poison arrow frogs, this substitution appeared to have no detrimental effect on acetylcholine binding and, thus, appeared cost-free. Results: The introduction of S108C into the α4ß2 nAChRs of non-dendrobatid frogs also does not affect ACh sensitivity, when these receptors are expressed in Xenopus laevis oocytes. However, α4ß2 nAChRs with C108 had a decreased magnitude of neurotransmitter-induced currents in all species tested ( Epipedobates anthonyi , non-dendrobatid frogs, as well as human), compared with α4ß2 nAChRs with the conserved S108. Immunolabeling of frog or human α4ß2 nAChRs in the plasma membrane using radiolabeled antibody against the ß2 nAChR subunit shows that C108 significantly decreased the number of cell-surface α4ß2 nAChRs, compared with S108. Conclusions: While S108C protects these species against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4ß2 nAChR function. These results may explain the high conservation of a serine at this site in vertebrates, as well as provide an example of a tradeoff between beneficial and deleterious effects of an evolutionary change. They also provide important clues for future work on assembly and trafficking of this important neurotransmitter receptor.

(+)-Catharanthine potentiates the GABAA receptor by binding to a transmembrane site at the ß(+)/α(-) interface near the TM2-TM3 loop.

(+)-Catharanthine, a coronaridine congener, potentiates the γ-aminobutyric acid type A receptor (GABAAR) and induces sedation through a non-benzodiazepine mechanism, but the specific site of action and intrinsic mechanism have not beendefined. Here, we describe GABAAR subtype selectivity and location of the putative binding site for (+)-catharanthine using electrophysiological, site-directed mutagenesis, functional competition, and molecular docking experiments. Electrophysiological and in silico experiments showed that (+)-catharanthine potentiates the responses to low, subsaturating GABA at ß2/3-containing GABAARs 2.4-3.5 times more efficaciously than at ß1-containing GABAARs. The activity of (+)-catharanthine is reduced by the ß2(N265S) mutation that decreases GABAAR potentiation by loreclezole, but not by the ß3(M286C) or α1(Q241L) mutations that reduce receptor potentiation by R(+)-etomidate or neurosteroids, respectively. Competitive functional experiments indicated that the binding site for (+)-catharanthine overlaps that for loreclezole, but not those for R(+)-etomidate or potentiating neurosteroids. Molecular docking experiments suggested that (+)-catharanthine binds at the ß(+)/α(-) intersubunit interface near the TM2-TM3 loop, where it forms H-bonds with ß2-D282 (TM3), ß2-K279 (TM2-TM3 loop), and ß2-N265 and ß2-R269 (TM2). Site-directed mutagenesis experiments supported the in silico results, demonstrating that the K279A and D282A substitutions, that lead to a loss of H-bonding ability of the mutated residue, and the N265S mutation, impair the gating efficacy of (+)-catharanthine. We infer that (+)-catharanthine potentiates the GABAAR through several H-bond interactions with a binding site located in the ß(+)/α(-) interface in the transmembrane domain, near the TM2-TM3 loop, where it overlaps with loreclezole binding site.

Modulation of α1ß3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix.

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a ß-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABAA α1ß3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the ß+α- interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in ß3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [ß3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1ß3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the ß+α- interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to ß3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the ß+α- interface for the anesthetic propofol.

Apremilast regulates acute effects of ethanol and other GABAergic drugs via protein kinase A-dependent signaling.

Phosphodiesterase type 4 (PDE4) inhibitors prevent hydrolysis of cyclic adenosine monophosphate and increase protein kinase A (PKA)-mediated phosphorylation. PDE4 inhibitors also regulate responses to ethanol and GABAergic drugs. We investigated mechanisms by which the PDE4 inhibitor, apremilast, regulates acute effects of ethanol and GABAergic drugs in male and female mice. Apremilast prolonged the sedative-hypnotic effects of gaboxadol, zolpidem, and propofol but did not alter etomidate effects, and unexpectedly shortened the sedative-hypnotic effects of diazepam. Apremilast prolonged rotarod ataxia induced by zolpidem, propofol, and loreclezole, shortened recovery from diazepam, but had no effect on ataxia induced by gaboxadol or etomidate. The PKA inhibitor H-89 blocked apremilast's ability to prolong the sedative-hypnotic effects of ethanol, gaboxadol, and propofol and to prolong ethanol- and propofol-induced ataxia. H-89 also blocked apremilast's ability to shorten the sedative-hypnotic and ataxic effects of diazepam. The ß1-specific antagonist, salicylidene salicylhydrazide (SCS), produced faster recovery from ethanol- and diazepam-induced ataxia, but did not alter propofol- or etomidate-induced ataxia. SCS shortened the sedative-hypnotic effects of ethanol and diazepam but not of propofol. In Xenopus oocytes, a phosphomimetic (aspartate) mutation at the PKA phosphorylation site in ß1 subunits decreased the maximal GABA current in receptors containing α1 or α3, but not α2 subunits. In contrast, phosphomimetic mutations at PKA sites in ß3 subunits increased the maximal GABA current in receptors containing α1 or α2, but not α3 subunits. The GABA potency and allosteric modulation by ethanol, propofol, etomidate, zolpidem, flunitrazepam, or diazepam were not altered by these mutations. We propose a model whereby apremilast increases PKA-mediated phosphorylation of ß1-and ß3-containing GABAA receptors and selectively alters acute tolerance to ethanol and GABAergic drugs.

Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance.

Animals that wield toxins face self-intoxication. Poison frogs have a diverse arsenal of defensive alkaloids that target the nervous system. Among them is epibatidine, a nicotinic acetylcholine receptor (nAChR) agonist that is lethal at microgram doses. Epibatidine shares a highly conserved binding site with acetylcholine, making it difficult to evolve resistance yet maintain nAChR function. Electrophysiological assays of human and frog nAChR revealed that one amino acid replacement, which evolved three times in poison frogs, decreased epibatidine sensitivity but at a cost of acetylcholine sensitivity. However, receptor functionality was rescued by additional amino acid replacements that differed among poison frog lineages. Our results demonstrate how resistance to agonist toxins can evolve and that such genetic changes propel organisms toward an adaptive peak of chemical defense.

Novel Molecule Exhibiting Selective Affinity for GABAA Receptor Subtypes.

Aminoquinoline derivatives were evaluated against a panel of receptors/channels/transporters in radioligand binding experiments. One of these derivatives (DCUK-OEt) displayed micromolar affinity for brain γ-aminobutyric acid type A (GABAA) receptors. DCUK-OEt was shown to be a positive allosteric modulator (PAM) of GABA currents with α1ß2γ2, α1ß3γ2, α5ß3γ2 and α1ß3δ GABAA receptors, while having no significant PAM effect on αß receptors or α1ß1γ2, α1ß2γ1, α4ß3γ2 or α4ß3δ receptors. DCUK-OEt modulation of α1ß2γ2 GABAA receptors was not blocked by flumazenil. The subunit requirements for DCUK-OEt actions distinguished DCUK-OEt from other currently known modulators of GABA function (e.g., anesthetics, neurosteroids or ethanol). Simulated docking of DCUK-OEt at the GABAA receptor suggested that its binding site may be at the α + ß- subunit interface. In slices of the central amygdala, DCUK-OEt acted primarily on extrasynaptic GABAA receptors containing the α1 subunit and generated increases in extrasynaptic "tonic" current with no significant effect on phasic responses to GABA. DCUK-OEt is a novel chemical structure acting as a PAM at particular GABAA receptors. Given that neurons in the central amygdala responding to DCUK-OEt were recently identified as relevant for alcohol dependence, DCUK-OEt should be further evaluated for the treatment of alcoholism.

Mutation of the inhibitory ethanol site in GABAA ρ1 receptors promotes tolerance to ethanol-induced motor incoordination.

Genes encoding the ρ1/2 subunits of GABAA receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6'Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6'Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (ß-alanine and taurine). We generated ρ1 (T6'Y) knockin (KI) mice using CRISPR/Cas9 to test the behavioral importance of the inhibitory actions of ethanol on this receptor. Both ρ1 KI and knockout (KO) mice showed faster recovery from acute ethanol-induced motor incoordination compared to WT mice. Both KI and KO mutant strains also showed increased tolerance to motor impairment produced by ethanol. The KI mice did not differ from WT mice in other behavioral actions, including ethanol intake and preference, conditioned taste aversion to ethanol, and duration of ethanol-induced loss of righting reflex. WT and KI mice did not differ in levels of ρ1 or ρ2 mRNA in cerebellum or in ethanol clearance. Our findings indicate that the inhibitory site for ethanol in GABAA ρ1 receptors regulates acute functional tolerance to moderate ethanol intoxication. We note that low sensitivity to alcohol intoxication has been linked to risk for development of alcohol dependence in humans.

Identification of an Inhibitory Alcohol Binding Site in GABAA ρ1 Receptors.

Alcohols inhibit γ-aminobutyric acid type A ρ1 receptor function. After introducing mutations in several positions of the second transmembrane helix in ρ1, we studied the effects of ethanol and hexanol on GABA responses using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. The 6' mutations produced the following effects on ethanol and hexanol responses: small increase or no change (T6'M), increased inhibition (T6'V), and small potentiation (T6'Y and T6'F). The 5' mutations produced mainly increases in hexanol inhibition. Other mutations produced small (3' and 9') or no changes (2' and L277 in the first transmembrane domain) in alcohol effects. These results suggest an inhibitory alcohol binding site near the 6' position. Homology models of ρ1 receptors based on the X-ray structure of GluCl showed that the 2', 5', 6', and 9' residues were easily accessible from the ion pore, with 5' and 6' residues from neighboring subunits facing each other; L3' and L277 also faced the neighboring subunit. We tested ethanol through octanol on single and double mutated ρ1 receptors [ρ1(I15'S), ρ1(T6'Y), and ρ1(T6'Y,I15'S)] to further characterize the inhibitory alcohol pocket in the wild-type ρ1 receptor. The pocket can only bind relatively short-chain alcohols and is eliminated by introducing Y in the 6' position. Replacing the bulky 15' residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short-chain alcohols. In conclusion, the net alcohol effect on the ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.

GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ1" antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ2" antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

GABA(A) receptor transmembrane amino acids are critical for alcohol action: disulfide cross-linking and alkyl methanethiosulfonate labeling reveal relative location of binding sites.

Alcohols and inhaled anesthetics modulate GABA(A) receptor (GABA(A)R) function via putative binding sites within the transmembrane regions. The relative position of the amino acids lining these sites could be either inter- or intra-subunit. We introduced cysteines in relevant TM locations and tested the proximity of cysteine pairs using oxidizing and reducing agents to induce or break disulfide bridges between cysteines, and thus change GABA-mediated currents in wild-type and mutant α1ß2γ2 GABA(A)Rs expressed in Xenopus laevis oocytes. We tested for: (i) inter-subunit cross-linking: a cysteine located in α1TM1 [either α1(Q229C) or α1(L232C)] was paired with a cysteine in different positions of ß2TM2 and TM3; (ii) intra-subunit cross-linking: a cysteine located either in ß2TM1 [ß2(T225C)] or in TM2 [ß2(N265C)] was paired with a cysteine in different locations along ß2TM3. Three inter-subunit cysteine pairs and four intra-subunits cross-linked. In three intra-subunit cysteine combinations, the alcohol effect was reduced by oxidizing agents, suggesting intra-subunit alcohol binding. We conclude that the structure of the alcohol binding site changes during activation and that potentiation or inhibition by binding at inter- or intra-subunit sites is determined by the specific receptor and ligand.

Functional validation of virtual screening for novel agents with general anesthetic action at ligand-gated ion channels.

GABA(A) receptors play a crucial role in the actions of general anesthetics. The recently published crystal structure of the general anesthetic propofol bound to Gloeobacter violaceus ligand-gated ion channel (GLIC), a bacterial homolog of GABA(A) receptors, provided an opportunity to explore structure-based ligand discovery for pentameric ligand-gated ion channels (pLGICs). We used molecular docking of 153,000 commercially available compounds to identify molecules that interact with the propofol binding site in GLIC. In total, 29 compounds were selected for functional testing on recombinant GLIC, and 16 of these compounds modulated GLIC function. Active compounds were also tested on recombinant GABA(A) receptors, and point mutations around the presumed binding pocket were introduced into GLIC and GABA(A) receptors to test for binding specificity. The potency of active compounds was only weakly correlated with properties such as lipophilicity or molecular weight. One compound was found to mimic the actions of propofol on GLIC and GABA(A), and to be sensitive to mutations that reduce the action of propofol in both receptors. Mutant receptors also provided insight about the position of the binding sites and the relevance of the receptor's conformation for anesthetic actions. Overall, the findings support the feasibility of the use of virtual screening to discover allosteric modulators of pLGICs, and suggest that GLIC is a valid model system to identify novel GABA(A) receptor ligands.

Alcohol Dependence and Genes Encoding α2 and γ1 GABAA Receptor Subunits: Insights from Humans and Mice.

One approach to identifying the causes of alcoholism, particularly without crossing ethical boundaries in human subjects, is to look at the person's genome (and particularly at the variations that naturally arise in the DNA) to identify those variations that seem to be found more commonly in people with the disease. Some of these analyses have focused on the genes that encode subunits of the receptor for the brain chemical (i.e., neurotransmitter) γ-aminobutyric acid (GABA). Different epidemiological genetic studies have provided evidence that variations in certain GABAA receptor (GABAA-R) subunits, particularly subunits α2 and γ1, are correlated with alcohol dependence. Manipulations of these genes and their expression in mice and rats also are offering clues as to the role of specific GABAA-Rs in the molecular mechanisms underlying alcoholism and suggest possibilities for new therapeutic approaches.

Mutations M287L and Q266I in the glycine receptor α1 subunit change sensitivity to volatile anesthetics in oocytes and neurons, but not the minimal alveolar concentration in knockin mice.

BACKGROUND: Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal glycine receptors with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement. METHODS: We produced single amino acid mutations into the glycine receptor α1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brainstem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice. RESULTS: Studies of glycine-evoked currents in brainstem neurons from knockin mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics. CONCLUSION: Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs.

Characterization of two mutations, M287L and Q266I, in the α1 glycine receptor subunit that modify sensitivity to alcohols.

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).

A transmembrane amino acid in the GABAA receptor ß2 subunit critical for the actions of alcohols and anesthetics.

Alcohols and inhaled anesthetics enhance the function of GABA(A) receptors containing α, ß, and γ subunits. Molecular analysis has focused on the role of the α subunits; however, there is evidence that the ß subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the α subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABA(A) receptor ß(2) subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant α(1)ß(2)(N265C)γ(2S) receptors [but not α(1)ß(2)(I264C)γ(2S)], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the ß(2) subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant α(1)ß(2)(N265C)γ(2S) receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.

Effects of acamprosate on neuronal receptors and ion channels expressed in Xenopus oocytes.

BACKGROUND: Acamprosate (calcium acetylhomotaurinate) has proven to be a moderately effective pharmacological adjunct for the treatment of alcoholism. However, the central nervous system mechanism by which acamprosate reduces alcohol relapse remains unclear. Here we survey a number of metabotropic receptors, ligand-gated ion channels, and voltage-gated ion channels, to determine if acamprosate has actions at these sites in the central nervous system. METHODS: Xenopus oocytes were injected with cDNAs or cRNAs encoding metabotropic glutamate receptors 1 and 5, M1 muscarinic receptors, glycine alpha1 homomeric and alpha1beta1 heteromeric receptors, gamma-aminobutyric acid A (GABA(A)alpha4beta3delta, alpha4beta3gamma2s, and alpha1beta2gamma2s) receptors, vanilloid receptor 1, and various combinations of alpha and beta subunits of voltage-gated Na+ channels. Electrophysiological responses were measured using two-electrode voltage clamp parameters after activation with agonists or voltage steps (for the voltage-gated channels). Acamprosate (0.1 to 100 microM) was pre-applied for 1 minute, followed by co-application with agonist. Acamprosate was also applied with ethanol to determine if it altered ethanol responses at some of these receptors and channels. RESULTS: None of the receptors or ion channels responded to acamprosate alone. Acamprosate also failed to alter the activation of receptors or channels by agonists or after activation of voltage-gated channels. There was no effect of acamprosate on ethanol responses at GABA(A)alpha1beta2gamma2s receptors or Na+ channels. CONCLUSIONS: Acamprosate does not significantly modulate the function of these receptors and ion channels at clinically relevant concentrations. Thus, the clinical effectiveness of acamprosate in the treatment of alcoholism is not likely due to direct effects on these receptors or ion channels.

Studies of ethanol actions on recombinant delta-containing gamma-aminobutyric acid type A receptors yield contradictory results.

The gamma-aminobutyric acid type A receptors (GABAA-Rs) display a wide variety of subunit combinations. Drugs such as benzodiazepines have shown differential effects based on GABAA-R subunit composition. Actions of alcohols and volatile anesthetics generally do not vary markedly with subunit composition, with low concentrations of ethanol being poor modulators of these receptors. Recent studies showed alpha(4/6)- and delta-containing GABAA-Rs (located extrasynaptically and responsible for tonic currents in selective brain regions) presenting high sensitivity to low concentrations of ethanol, but these results have not been obtained in other laboratories. We carried out additional experiments varying the receptor level of expression, and GABA and ethanol concentration, but no sensitivity to low concentrations of ethanol was detected. We will discuss these results and attempt an analysis of the possible causes for the discrepancies.