1 A comparative study of the actions of structurally diverse allosteric modulators on mammalian (human alpha(3) beta(2) gamma(21)) 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. EC(50) values of 102+/-18 mu M; 152+/-10 mu M and 9.8+/-1.7 mu M were determined for human alpha(3) beta(1) gamma(21), 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 EC(10), the EC(50) for potentiation was estimated to be 45+/-1 mu M, 312+/-8 mu M and 837+/-25 mu M for human alpha(3) beta(1) gamma(21), Rdl splice variant and Rdl receptors respectively. Maximal enhancement (expressed relative to the current induced by the EC(10) 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 alpha(3) beta(1) gamma(21) receptor with an EC(50) of 1.2+/-0.03 mM and had a maximal effect amounting to 3.3+/-0.4 fold of the response evoked by the EC(10) concentration of GABA. Currents evoked by pentobarbitone were blocked by 10-30 mu M picrotoxin and potentiated by 0.3 mu M flunitrazepam. Pentobarbitone did not directly activate the invertebrate GABA receptors. 5 5 alpha-Pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents mediated by the human alpha(3) beta(1) gamma(21) receptor with an EC(50) of 87+/-3 nM and a maximal enhancement of 6.7+/-0.8 fold of that produced by the GABA EC(10) concentration. By contrast, relatively high concentrations (3-10 mu M) of this steroid had only a modest effect on the Rdl receptor and its splice variant. 6 A small direct effect of 5 alpha-pregnan-3 alpha-ol-20-one (0.3-10 mu M) was detected for the human alpha(3) beta(1) gamma(21) receptor (maximal effect only 0.08+/-0.01 times that of the GABA EC(10)). This response was antagonized by 30 mu M picrotoxin and enhanced by flunitrazepam (0.3 mu M). 5 alpha-Pregnan-3 alpha-ol-20-one did not directly activate the invertebrate GABA receptors. 7 Propofol enhanced GABA-evoked currents mediated by human alpha(3) beta(1) gamma(21) and Rdl splice variant receptors with EC(50) values of 3.5+/-0.1 mu M and 8+/-0.3 mu M 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 EC(10)). 8 Propofol directly activated the human alpha(3) beta(1) gamma(21) receptor with an EC(50) of 129+/-10 mu M, and at a maximally effective concentration, evoked a current amounting to 3.5+/-0.5 times that elicited by a concentration of GABA producing 10% of the maximal response. The response to propofol was blocked by 10-30 mu M picrotoxin and enhanced by flunitrazepam (0.3 mu M). Propofol did not directly activate the invertebrate Rdl splice variant receptor. 9 GABA-evoked currents mediated by the human alpha(3) beta(1) gamma(21) receptor were potentiated by etomidate (EC(50) = 7.7+/-0.2 mu M) and maximally enhanced to 8+/-0.8 fold of the response to an EC(10) concentration of GABA. By contrast, the Rdl, or Rdl splice variant forms of the invertebrate GABA receptor were insensitive to the positive allosteric modulating actions of etomidate. Neither the mammalian nor the invertebrate receptors, were directly activated by etomidate. 10 delta-Hexachlorocyclohexane enhanced GABA-evoked currents with EC(50) values of 3.4+/-0.1 mu M and 3.0+/-0.1 mu M for the human alpha(3) beta(1) gamma(21) receptor and the Rdl splice variant receptor respectively. The maximal enhancement was 4.5+/-0.3 and 10.3+/-0.3 fold that produced by the appropriate EC(10) concentration of GABA for the mammalian and invertebrate receptors respectively. delta-Hexachlorocyclohexane did not directly activate either receptor type. 11 Loreclezole potentiated GABA-evoked currents with an EC(50) of 7.4+/-0.2 mu M and 20+/-1 mu M for the human alpha(3) beta(1) gamma(21) and Rdl splice variant receptors respectively. A maximal enhancement of 1.9+/-0.2 and 6.9+/-0.2 fold (relative to the response produced by an EC(10) concentration of GABA) was found for the mammalian and invertebrate receptors respectively. Loreclezole did not directly activate either receptor type. 12 Both the invertebrate Rdl receptor and its splice variant function efficiently as homo-oligomeric complexes upon expression in Xenopus laevis oocytes. This feature, combined with the differential pharmacology of the invertebrate and human receptors towards a variety of positive allosteric modulators, may be useful in future studies designed to determine drug binding domains on the receptor protein.
