Zinc-dependent modulation of α2- and α3-glycine receptor subunits by ethanol.

BACKGROUND: Strychnine-sensitive glycine receptors (GlyRs) are expressed throughout the brain and spinal cord and are among the strongly supported protein targets of alcohol. This is based largely on studies of the α1-subunit; however, α2- and α3-GlyR subunits are as or more abundantly expressed than α1-GlyRs in multiple forebrain brain areas considered to be important for alcohol-related behaviors, and uniquely some α3-GlyRs undergo RNA editing. Nanomolar and low micromolar concentrations of zinc ions potentiate GlyR function, and in addition to zinc's effects on glycine-activated currents, we have recently shown that physiological concentrations of zinc also enhance the magnitude of ethanol (EtOH)'s effects on α1-GlyRs. METHODS: Using 2-electrode voltage-clamp electrophysiology in oocytes expressing either α2- or α3-GlyRs, we first tested the hypothesis that the effects of EtOH on α2- and α3-GlyRs would be zinc dependent, as we have previously reported for α1-GlyRs. Next, we constructed an α3P185L-mutant GlyR to test whether RNA-edited and unedited GlyRs contain differences in EtOH sensitivity. Last, we built a homology model of the α3-GlyR subunit. RESULTS: The effects of EtOH (20 to 200 mM) on both subunits were greater in the presence than in the absence of 500 nM added zinc. The α3P185L-mutation that corresponds to RNA editing increased sensitivity to glycine and decreased sensitivity to EtOH. CONCLUSIONS: Our findings provide further evidence that zinc is important for determining the magnitude of EtOH's effects at GlyRs and suggest that by better understanding zinc/EtOH interactions at GlyRs, we may better understand the sites and mechanisms of EtOH action.

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.

Male and female mice develop escalation of heroin intake and dependence following extended access.

Opioid use disorder is a serious public health issue in the United States. Animal models of opioid dependence are fundamental for studying the etiology of addictive behaviors. We tested the hypothesis that extended access to heroin self-administration leads to increases in heroin intake and produces somatic signs of opioid dependence in both male and female mice. Adult C57BL/6J mice were trained to nosepoke (fixed-ratio 1) to obtain intravenous heroin in six daily 1-h sessions (30-60 µg/kg/infusion). The mice were divided into short access (ShA; 1 h) and long access (LgA; 6 h) groups. Immediately after the 10th escalation session, the mice received a challenge dose of naloxone (1 mg/kg), and somatic signs of withdrawal were recorded. The mice readily acquired intravenous heroin self-administration. LgA mice escalated their drug intake in the first hour across sessions and had significantly higher scores of somatic signs of naloxone-precipitated opioid withdrawal compared with ShA mice. Female mice exhibited increases in heroin intake compared with male mice. Male and female mice exhibited similar levels of somatic signs of withdrawal. Because of the wide availability of genetically modified mouse lines, the present mouse model may be particularly useful for better understanding genetic and sex differences that underlie the transition to compulsive-like opioid taking and seeking.

Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 µM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model for alcohol/anesthetic interaction with human GLRA1.