Hormonally regulated alpha(4)beta(2)delta GABA(A) receptors are a target for alcohol

Abstract

Here we report that low concentrations of alcohol (1-3 mM) increased Cl(-) currents gated by a recombinant GABA(A) receptor, alpha(4)beta(2)delta, by 40-50% in Xenopus laevis oocytes. We also found greater hippocampal expression of receptors containing alpha(4) and delta subunits, using a rat model of premenstrual syndrome (PMS) in which 1-3 mM alcohol preferentially enhanced GABA-gated currents, and low doses of alcohol attenuated anxiety and behavioral reactivity. The alcohol sensitivity of delta-containing receptors may underlie the reinforcing effects of alcohol during PMS, when eye saccade responses to low doses of alcohol are increased.

Fig. 1

Low concentrations of alcohol potentiate GABA responses at α₄β₂δ receptors. (a) The effects of alcohol on responses to GABA (EC₂₀ = 0.03 μM α₄β₂δ, 1.7 μM α₁β₂γ_2s, 5.6 μM α₄β₂γ_2s) were measured by two-electrode voltage-clamp recording at −70 mV in oocytes expressing recombinant GABA_A receptors. Inset, representative traces showing currents activated by GABA in the absence and presence of 1 mM alcohol. (b) Effects of 1 mM alcohol on GABA(EC₂₀)-gated currents at various GABA_A receptor subtypes (n = 8–20, *P < 0.05). Experiments were conducted according to Institutional Animal Care and Use Committee guidelines.

Fig. 2

Progesterone withdrawal increases α₄βδ GABA_A receptors. (a) Left, western blot showing increased expression of the δ subunit (54 kDa), but not a control protein (GAPDH, 36 kDa) after progesterone withdrawal (P Wd) compared to control (Con). Right, mean values (n = 20–25, performed in triplicate). (b) Increases in α₄ protein following P Wd were prevented by in vivo administration of alcohol (0.5 g/kg × 3, intraperitoneally) during the final two hours of the withdrawal period (P Wd + Alc; n = 9–10). (c) Co-assembly of α₄ and δ GABA_A receptor subunits. After immunoprecipitation (IP) using protein A beads coupled to antibodies for the δ subunit (IP, δ) or a cytosolic protein (IP, Neg. con) membranes were probed with digoxygenin-labeled anti-α₄ on a western blot (n = 4 hippocampi, in duplicate). A prominent 67-kDa band was detected after P Wd, but was barely detectable under control conditions. (d) The maximum current produced by THIP compared to that produced by 10 mM GABA was 1.41 after P Wd (THIP EC₅₀ = 39 ± 2 μM), and 0.95 in control neurons (THIP EC₅₀ = 81 ± 6 μM). This was determined using whole-cell patch clamp recording in neurons from CA1 hippocampus (n = 10–15, *P < 0.05).

Fig. 3

Low doses of alcohol potentiate GABA-gated currents in hippocampal pyramidal neurons and decrease behavioral excitability after progesterone withdrawal. (a) GABA (10 μM)-gated currents recorded in the presence or absence of alcohol (0.1–10 mM), measured by whole-cell patch clamp recording after progesterone withdrawal (P Wd) or under control conditions (con). Inset, representative traces. Suppression of α₄ subunit expression (P Wd + α₄ suppressed), as described in Fig. 2b, abolished the stimulatory effects of low concentrations of alcohol after P Wd (n = 20–25 cells/concentration, 8–10 rats/group). (b) Low doses of alcohol administered to P Wd (but not control) rats significantly lowered the acoustic startle response, expressed relative to the saline control (n = 5–11, *P < 0.05, **P < 0.005).