Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes

1. A comparative study of the actions of structurally diverse allosteric modulators on mammalian (human α₃ β₂ γ(2L)) or invertebrate (Drosophila melanogaster Rdl or a splice variant of Rdl) recombinant GABA receptors has been made using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. 2. Oocytes preinjected with the appropriate cRNAs responded to bath applied GABA with a concentration-dependent inward current. EC₅₀ values of 102 ± 18 μM; 152 ± 10 μM and 9.8 ± 1.7 μM were determined for human α₃ β₁ γ(2L), Rdl splice variant and the Rdl receptors respectively. 3. Pentobarbitone enhanced GABA-evoked currents mediated by either the mammalian or invertebrate receptors. Utilizing the appropriate GABA EC₁₀, the EC₅₀ for potentiation was estimated to be 45 ± 1 μM, 312 ± 8 μM and 837 ± 25 μM for human α₃ β₁ γ(2L), Rdl splice variant and Rdl receptors respectively. Maximal enhancement (expressed relative to the current induced by the EC₁₀ concentration of GABA where this latter response = 1) at the mammalian receptor (10.2 ± 1 fold) was greater than that at either the Rdl splice variant (5.5 ± 1.3 fold) or Rdl (7.9 ± 0.8 fold) receptors. 4. Pentobarbitone directly activated the human α₃ β₁ γ(2L) receptor with an EC₅₀ of 1.2 ± 0.03 mM and had a maximal effect amounting to 3.3 ± 0.4 fold of the response evoked by the EC₁₀ concentration of GABA. Currents evoked by pentobarbitone were blocked by 10-30 μM picrotoxin and potentiated by 0.3 μM flunitrazepam. Pentobarbitone did not directly activate the invertebrate GABA receptors. 5. 5α-Pregnan-3α-ol-20-one potentiated GABA-evoked currents mediated by the human α₃ β₁ γ(2L) receptor with an EC₅₀ of 87 ± 3 nM and a maximal enhancement of 6.7 ± 0.8 fold of that produced by the GABA EC₁₀ concentration. By contrast, relatively high concentrations (3-10 μM) of this steroid had only a modest effect on the Rdl receptor and its splice variant. 6. A small direct effect of 5α-pregnan-3α-ol-20-one (0.3-10 μM) was detected for the human α₃ β₁ γ(2L) receptor (maximal effect only 0.08 ± 0.01 times that of the GABA EC₁₀). This response was antagonized by 30 μM picrotoxin and enhanced by flunitrazepam (0.3 μM). 5α-Pregnan-3α-ol-20-one did not directly activate the invertebrate GABA receptors. 7. Propofol enhanced GABA-evoked currents mediated by human α₃ β₁ γ(2L) and Rdl splice variant receptors with EC₅₀ values of 3.5 ± 0.1 μM and 8 ± 0.3 μM respectively. The maximal enhancement was similar at the two receptor types (human 11 ± 1.8 fold; invertebrate 8.8 ± 1.4 fold that of the GABA EC₁₀).