Low dose acute alcohol effects on GABA A receptor subtypes

Abstract

GABA(A) receptors (GABA(A)Rs) are the main inhibitory neurotransmitter receptors and have long been implicated in mediating at least part of the acute actions of ethanol. For example, ethanol and GABAergic drugs including barbiturates and benzodiazepines share many pharmacological properties. Besides the prototypical synaptic GABA(A)R subtypes, nonsynaptic GABA(A)Rs have recently emerged as important regulators of neuronal excitability. While high doses (> or =100 mM) of ethanol have been reported to enhance activity of most GABA(A)R subtypes, most abundant synaptic GABA(A)Rs are essentially insensitive to ethanol concentrations that occur during social ethanol consumption (< 30 mM). However, extrasynaptic delta and beta3 subunit-containing GABA(A)Rs, associated in the brain with alpha4 or alpha6 subunits, are sensitive to low millimolar ethanol concentrations, as produced by drinking half a glass of wine. Additionally, we found that a mutation in the cerebellar alpha6 subunit (alpha6R100Q), initially reported in rats selectively bred for increased alcohol sensitivity, is sufficient to produce increased alcohol-induced motor impairment and further increases of alcohol sensitivity in recombinant alpha6beta3delta receptors. Furthermore, the behavioral alcohol antagonist Ro15-4513 blocks the low dose alcohol enhancement on alpha4/6/beta3delta receptors, without reducing GABA-induced currents. In binding assays alpha4beta3delta GABA(A)Rs bind [(3)H]Ro15-4513 with high affinity, and this binding is inhibited, in an apparently competitive fashion, by low ethanol concentrations, as well as analogs of Ro15-4513 that are active to antagonize ethanol or Ro15-4513's block of ethanol. We conclude that most low to moderate dose alcohol effects are mediated by alcohol actions on alcohol/Ro15-4513 binding sites on GABA(A)R subtypes.

Fig. 1

A fraction of cultured neurons are enhanced by low doses of ethanol. (A) Reversible enhancement of GABA currents, evoked by application of 2.5 μM GABA, in whole cell voltage clamped (holding potential, 50 mV) cerebral cortical neurons by 20 mM ethanol. (B) Shows a neuron with EtOH-insensitive (20 mM) GABA currents that are enhanced by flurazepam (20 μM) and pentobarbital (20 μM). (Figure from Aguayo, 1990; with permission from the author and the publisher.)

Fig. 2

A point mutation in the cerebellar α6 subunit leads to increased alcohol sensitivity on (A) recombinant receptors expressed in oocytes, (B) in animals carrying this mutation and (C) in tonic currents from cerebellar granule cells. (A) Currents were recorded from oocytes with either α6β3δ or α6R100Qβ3δ mutant receptors. Ethanol, (concentrations in mM) co-applied with 300 nM GABA, leads to a dose-dependent and reversible increase in holding current. Receptors containing the α6R100Q mutation show a dramatically increased EtOH response. (B) Rats homozygous for either the α6-100R or α6-100Q allele were tested on an accelerating rotarod (4–40 rpm within 5 min) before, and 20, 40 and 60 min after intraperitoneal ethanol (or saline) injection. Under control conditions (saline injection) these groups of rats do not differ. However, after a low dose of EtOH (0.75 g/kg i.p.) where rats carrying the α6-100R allele (n = 7) show no signs of motor impairment, α6-100QQ (n = 8) rats are already significantly impaired. Error bars (SEM) do not, or only slightly, exceed the symbol sizes. (C) Tonic GABA current recorded from cerebellar granules cells in slices from α6-100RR (upper traces) and α6-100QQ (lower traces) rats show that the α6-100Q mutation leads to a dramatic increase in ethanol (30 and 100 mM) enhancement of the tonic current. (Figure reproduced from Hanchar et al., 2005).

Fig. 3

Alcohol enhancement of GABA_ARs by 50 mM (or 30) EtOH in (A)Cl⁻ flux assays, (B) in neurons, and (C) in recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist Ro15-4513. (A) Left panel shows that the dose-dependent block of 50 mM ethanol enhancement of ³⁶Cl⁻ uptake into rat cortical synaptoneurosomes by Ro15-4513, with complete block at 100 nM. The right panel shows percent stimulation of ³⁶Cl⁻ flux by EtOH alone (●) or in the presence of 100 nM Ro15-4513 (○). (Figure from Suzdak et al., 1986a.) (B) GABA currents were evoked by brief pressure pulses of GABA (25 μM GABA in the pipette) to the soma of a cultured rat cerebral cortical neurons. The resulting GABA current is enhanced by bath application of 50 mM ethanol. In the same cell the EtOH enhancement is blocked by bath co-application 100 nM Ro15-4513 together with 50 mM ethanol (right panel). (Figure redrawn from Reynolds et al., 1992, with permission from Elsevier.) (C) Recombinant α4β3δ GABA_AR are enhanced by 30 mM ethanol and this enhancement is blocked by 100 nM Ro15-4514. The ethanol blocking effects are reversed by flumazenil (Ro15-1788) as well as β-CCE (each at 300 nM), but not by the classical benzodiazepine flunitrazepam or the β-carboline DMCM (each at 1 μM). Note that both flumazenil as well as β-CCE have been reported to reverse the alcohol antagonism observed with Ro15-4513 rats. (Figure modified from Wallner et al., 2006.)

Fig. 4

Native immuno-purified and recombinant δ subunit-containing receptors contain a high affinity [³H]Ro15-4513 binding site that is sensitive to alcohol displacement. (A) Left panel: [³H]Ro15-4513 binding (10 nM) to δ subunit-antibody immuno purified cerebellar and recombinant α4β3δ GABA_AR is inhibited by low ethanol concentrations. In contrast, [³H]Ro15-4513 binding to classical γ2 subunit-containing receptors is not altered in the presence of ethanol. Right panel: Pharmacological characterization of the [³H]Ro15-4513 binding site on α4β3δ receptors. Compounds that have been previously shown to prevent Ro15-4513 antagonism (Ro15-1788, β-CCE, FG7142) or that show behavioral alcohol antagonism (RY024, RY080) displace [³H]Ro15-4513 from its binding site on α4β3δ receptors. Classical benzodiazepine agonists like diazepam, midazolam or flunitrazepam do not displace [³H]Ro15-4513 from α4β3δ GABA_ARs at concentrations <100 μM. (B) Chemical structures of ethanol and the imidazobenzodiazepines Ro15-4513 and flumazenil. Compounds RY080 and RY024 are congeners of Ro15- 4513 that carry an acetylene group in place of the azido group in Ro15-4513. (Figure reproduced from Hanchar et al., 2006.)

Fig. 5

Immunolocalization of the GABA_AR subunits (α4, α6, β3, δ) that form highly alcohol-sensitive GABA_AR subtypes. The α6 subunit is exclusively expressed in the cerebellar granule cells (CG), the most numerous neuron in the brain that also has high levels of β3 and δ subunit protein. Receptors formed by α6βδ subunits are known to mediate tonic currents in cerebellar granule cells and alcohol effects on these receptors make an important contribution to alcohol-induced motor impairment (Hanchar et al., 2005). Receptors formed by α4 and δ subunits are more widespread in the brain and are responsible for tonic currents, for example, in dentate gyrus granule (DGG) neurons in the hippocampus. Like α4β3δ recombinant receptors expressed in oocytes, tonic currents in DGG cells are highly sensitive to ethanol (Wei et al., 2004). Both CG and DGG neurons show high sensitivity to the neuroactive steroid THDOC (Stell et al., 2003). (Figure reproduced from Pirker et al., 2000, with permission from Elsevier.)