Selected channel-lining cysteine mutants from the M2 segment of rat ␣1 ␥-aminobutyric acid (GABA) type A receptor subunit, at positions 257, 261, 264, and 272 were co-expressed with ␤1 and ␥2 subunits in Xenopus oocytes. They generated functional receptors displaying conductance and response to both GABA and picrotoxinin similar to the wild type ␣1␤1␥2 receptor. Three chemically reactive affinity probes derived from noncompetitive blockers were synthesized to react with the engineered cysteines: 1) dithiane bis-sulfone derivative modified by an isothiocyanate function (probe A); 2) fiprole derivatives modified by an ␣-chloroketone (probe B) and ␣-bromoketone (probe C) moiety. These probes blocked the GABA-induced currents on all receptors. This blockade could be fully reversed by a washing procedure on the wild type, the ␣1T261C␤1␥2 and ␣1L264C␤1␥2 mutant receptors. In contrast, an irreversible effect was observed for all three probes on both ␣1V257C␤1␥2 and ␣1S272C␤1␥2 mutant receptors. This effect was probe concentration-dependent and could be abolished by picrotoxinin and/or t-butyl bicyclophosphorothionate. These data indicate a major interaction of non-competitive blockers at position 257 of the presumed M2 segment of rat ␣1 subunit but also suggest an interaction at the more extracellular position 272. ␥-Aminobutyric acid type A (GABA A ) 1 receptors exert their inhibitory effect in the central nervous system of vertebrates by regulating a chloride-sensitive channel which is very likely centered within a protein transmembrane heteropentameric subunits complex (1-5). The existence of 6␣, 4␤, 4␥, 1␦, and 2 subunits in addition to splicing variants, suggests a large diversity in the constitution of heteropentameric isoforms allowing a subtle tuning of the action of this neurotransmitter (6 -8). However, it has been proposed that a restricted number of combinations condition the functioning of this receptor and it is assumed that the ␣1␤2␥2 represents the major adult isoform (9). GABA A receptors serve as the target for several classes of molecules including important neuroactive drugs such as benzodiazepines, barbiturates, and neurosteroids. In contrast, only three receptor subunits have been cloned from insects up to now, RDL (10), ␤ (11), and GRD (12) leading to an apparently less complex situation for their structural assembly. Of particular interest are the action of non-competitive antagonists which are presumed to interact within the GABA receptor chloride channel leading to powerful insecticidal properties when presenting a selectivity for insect GABA receptor (13-16).To investigate, at a molecular level, the interaction of noncompetitive GABA antagonists with the chloride channel associated to the GABA A receptor, we defined an approach which uses chemically reactive non-competitive blockers (NCBs) as chemical sensors for cysteine mutants on the rat ␣1 GABA receptor subunit. This strategy was derived from the extensive work of Akabas and co-workers on several ionic channels including the chloride chan...

This paper reports the functional expression and pharmacological characterization of a full length complementary deoxyribonucleic acid (cDNA) (pIVY12) cloned from a Heliothis virescens fertilized egg cDNA library that encodes for a gamma-aminobutyric acid (GABA) receptor subunit (HVRDL-Ser 285). Two electrode voltage clamp recordings of Xenopus oocytes expressing the HVRDL GABA-gated chloride channel revealed robust chloride ion conductance in response to GABA and the GABAA receptor agonist, muscimol. Baclofen, a GABAB agonist had no effect. Phenobarbital showed a positive dose-dependent allosteric modulatory effect, whereas the benzodiazepine, flunitrazepam, had no effect. Chloride conductance was depressed by the novel insecticide, fipronil ((+/-)-5-amino-1-(2,6 dichloro-alpha, alpha, alpha-trifluoro-p-tolyl)-4-trifluoromethyl-sulfinylpyrazole-3-carb onitrile) and the GABAA antagonist, picrotoxinin. The HVRDL GABA receptor was insensitive to blockage by dieldrin and the GABAA antagonist, bicuculline. The comparative actions of fipronil, picrotoxinin and dieldrin were examined on oocytes expressing the H. virescens wild-type (HVRDL-Ser 285), the site-directed mutant (HVRDL-Ala 285), the Drosophila melanogaster Rdl wild-type (DMRDL-Ala 302) and the Rdl dieldrin resistant (DMRDL-Ser 302) homo-oligomeric GABA receptors. HVRDL-Ala 285 was 15-fold more sensitive to blockage by fipronil than HVRDL-Ser 285. DMRDL-Ala 302 and DMRDL-Ser-302 showed a similar level of sensitivity to blockage by fipronil. HVRDL-Ser 285 and DMRDL-Ser 302 exhibited a similar level of insensitivity to picrotoxinin. HVRDL-Ala 285 and DMRDL-Ala 302 showed a similar range of picrotoxinin sensitivity. DMRDL-Ala 302 and HVRDL-Ala 285 showed some sensitivity to blockage by dieldrin. Fipronil sensitivity was significantly altered by the serine to alanine mutation at position 285 in the M2 region of the HVRDL subunit, whereas no difference was observed between the DMRDL-Ser 302 and DMRDL-Ala 302 receptors.

No abstract

No abstract