Binding and Physiology of 4′-Ethynyl-4- n-propylbicycloorthobenzoate (EBOB) in Cyclodiene-Resistant Drosophila

Abstract

4′-Ethynyl-4- n-[2,3- 3H 2]propylbicycloorthobenzoate ([ 3H]EBOB) is a novel radioligand for the convulsant binding site of vertebrate and invertebrate γ-aminobutyric acid (GABA) receptors. Previous studies in membranes from house fly heads have shown [ 3H]EBOB to have high affinity for the cyclodiene binding site, which was reduced fourfold in cyclodiene-resistant strains. Following our recent identification of single amino acid replacements in the Drosophila GABA receptor gene Rdl conferring resistance to cyclodienes, we were interested in correlating [ 3H]EBOB binding and physiology with specific replacements of alanine302 in Rdl. Here we report that [ 3H]EBOB binding is not detectable in resistant strains carrying either the resistance-associated alanine302> serine ( Drosophila melanogaster or D. simulans) or the alanine302> glycine ( D. simulans) replacement. Thus, despite high specific binding to membranes from susceptible flies, no binding higher than nonspecific was observed in resistant preparations. EBOB (100 n M) was also shown to functionally block GABA-gated chloride ion currents generated in insect cells infected with a recombinant susceptible Rdl baculovirus, while cells expressing constructs containing the alanine302> serine replacement showed 10-fold insensitivity to block. These results indicate that the presence of alanine302 is central to the binding of [ 3H]EBOB to GABA receptors containing Rdl subunits and confirm the usefulness of this radioligand for the study of this important binding site.