DROSOPHILA GABA-GATED CHLORIDE CHANNEL - MODIFIED [H-3] EBOB BINDING-SITE ASSOCIATED WITH ALA-]SER OR GLY MUTANTS OF RDL SUBUNIT

The non-competitive blocker site of the GABA-gated chloride ion channe l in normal susceptible strains of Drosophila melanogaster and simulan s binds 4-n-[H-3]propyl-4'-ethynylbicycloorthobenzoate ([H-3]EBOB) spe cific sites with K(d)s of 1.6-1.9 nM and B(max)s of 171-181 fmol/mg pr otein. This specific binding of [H-3]EBOB is strongly inhibited by: a large number and variety of insecticidal channel blockers at 20 nM (li ndane, alpha-endosulfan, dieldrin, 12-ketoendrin, fipronil, and a repr esentative bicycloorthobenzoate and dithiane) or 200 nM (picrotoxinin) ; the insecticidal channel activators avermectin and moxidectin at 20 nM; muscimol at 30 mu M and GABA at 300 mu M. Cyclodiene resistance in D. melanogaster has been attributed to a mutation resulting in an Ala (302) --> Ser replacement in the Rdl GABA receptor subunit and in D. s imulans to an homologous Ala --> Ser or Gly replacement. These mutatio ns are shown here to greatly reduce [H-3]EBOB binding, i.e. lower affi nity and apparent number of binding sites. The Ala --> Ser replacement with both melanogaster and simulans almost always reduces the potency in inhibiting [H-3]EBOB binding of each of eight channel blockers and of muscimol and GABA. The Ala --> Gly replacement in D. simulans is g enerally less effective than the Ala --> Ser modification in reducing sensitivity to the channel blockers and to muscimol and GABA. The chan nel activators avermectin and moxidectin usually retain their inhibito ry potency in the Rdl subunit mutants. Thus, it appears that replaceme nt of the Ala by Ser generally modifies the non-competitive blocker si te and its coupling to the AGBA-recognition site with less effect on t he channel activator site. In contrast, the Ala --> Gly replacement ha s less impact in protecting the chloride channel from the action of in secticidal blockers. Each of the resistant strains has the same level of resistance to the lethal action of the five channel blockers examin ed but none to avermectins and muscimol.