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

Abstract

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.

Figure 1

Tracings showing the ethanol effect on EC₂₀ GABA responses (long application) in rat α₄β₃δ expressed in oocytes. The faster desensitization of the GABA current in rat α₄β₃γ_2S is shown for comparison. Reproduced by permission of The American Society for Pharmacology and Experimental Therapeutics, adapted from Borghese et al. (2006).

Figure 2

Tracings and graph showing the ethanol effect on EC₂₀ GABA responses (short application) in rat α₄β₃ (A), α₄β₃γ_2S (B) and α₄β₃δ (C) expressed in oocytes. (D) Pooled results; values are mean ± S.E.M., n= 6-8, * p< 0.05 versus other subunit combinations at 300 mM ethanol. Reproduced by permission of The American Society for Pharmacology and Experimental Therapeutics, adapted from Borghese et al. (2006).

Figure 3

GABA responses of rat subunits injected in different ratios in oocytes. GABA concentration-response curves (A) and GABA maximal responses 7-9 days after injection (B) when α₄β₃γ_2S (subunit ratio 1:1:3) and α₄β₃δ (subunit ratio ranging from 1:1:10 to 1:1:0.01) were expressed. In A, n=3-12; in B, *p<0.01 versus α₄β₃δ 1:1:10 (Dunnett’s Multiple Comparison Test, ANOVA), n=4-25.

Figure 4

GABA modulation by zinc and ethanol of rat subunits injected in different ratios in oocytes. Zinc (1 μM) inhibition (A) and ethanol (30 and 100 mM) potentiation (B) of EC₂₀ GABA responses when α₄β₃γ_2S (subunit ratio 1:1:3) and α₄β₃δ (subunit ratio ranging from 1:1:10 to 1:1:0.01) were expressed. *p< 0.01 (Dunnett’s Multiple Comparison Test, ANOVA), n= 3-8.

Figure 5

Modulation by ethanol of a lower concentration of GABA (EC₅) applied to rat α₄β₃δ and α₄β₃γ_2S expressed in oocytes. n= 4.

Figure 6

GABA modulation by zinc and ethanol of rat α₄β₃δ and α₄β₃γ_2S expressed in oocytes. (A) Zinc (0.01-1 μM) effect on EC₅ GABA responses. (B) Difference between the percentage of change in the EC₅ GABA response when coapplied with different concentrations of zinc (0-1 μM) and 30 mM ethanol, and when coapplied with zinc alone at those same concentrations. N= 4-5.