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 allosteri c modulators on mammalian (human alpha(3) beta(2) gamma(21)) or invert ebrate (Drosophila melanogaster Rdl or a splice variant of Rdl) recomb inant GABA receptors has been made using the Xenopus laevis oocyte exp ression system and the two electrode voltage-clamp technique. 2 Oocyte s preinjected with the appropriate cRNAs responded to bath applied GAB A 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 r espectively. 3 Pentobarbitone enhanced GABA-evoked currents mediated b y either the mammalian or invertebrate receptors. Utilizing the approp riate 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) ga mma(21), Rdl splice variant and Rdl receptors respectively. Maximal en hancement (expressed relative to the current induced by the EC(10) con centration of GABA where this latter response = 1) at the mammalian re ceptor (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 Pentobar bitone directly activated the human alpha(3) beta(1) gamma(21) recepto r with an EC(50) of 1.2+/-0.03 mM and had a maximal effect amounting t o 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 pi crotoxin and potentiated by 0.3 mu M flunitrazepam. Pentobarbitone did not directly activate the invertebrate GABA receptors. 5 5 alpha-Preg nan-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 G ABA EC(10) concentration. By contrast, relatively high concentrations (3-10 mu M) of this steroid had only a modest effect on the Rdl recept or 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) bet a(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 Pr opofol 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 b locked by 10-30 mu M picrotoxin and enhanced by flunitrazepam (0.3 mu M). Propofol did not directly activate the invertebrate Rdl splice var iant receptor. 9 GABA-evoked currents mediated by the human alpha(3) b eta(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 v ariant forms of the invertebrate GABA receptor were insensitive to the positive allosteric modulating actions of etomidate. Neither the mamm alian nor the invertebrate receptors, were directly activated by etomi date. 10 delta-Hexachlorocyclohexane enhanced GABA-evoked currents wit h EC(50) values of 3.4+/-0.1 mu M and 3.0+/-0.1 mu M for the human alp ha(3) beta(1) gamma(21) receptor and the Rdl splice variant receptor r espectively. 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-Hexachlorocy clohexane did not directly activate either receptor type. 11 Loreclezo le potentiated GABA-evoked currents with an EC(50) of 7.4+/-0.2 mu M a nd 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 an d 6.9+/-0.2 fold (relative to the response produced by an EC(10) conce ntration of GABA) was found for the mammalian and invertebrate recepto rs respectively. Loreclezole did not directly activate either receptor type. 12 Both the invertebrate Rdl receptor and its splice variant fu nction efficiently as homo-oligomeric complexes upon expression in Xen opus laevis oocytes. This feature, combined with the differential phar macology of the invertebrate and human receptors towards a variety of positive allosteric modulators, may be useful in future studies design ed to determine drug binding domains on the receptor protein.