BINDING AND PHYSIOLOGY OF 4'-ETHYNYL-4-N-PROPYLBICYCLOORTHOBENZOATE (EBOB) IN CYCLODIENE-RESISTANT DROSOPHILA

Ethynyl-4-n-[2,3-H-3(2)]propylbicycloorthobenzoate ([H-3]EBOB) is a no vel radioligand for the convulsant binding site of vertebrate and inve rtebrate gamma-aminobutyric acid (GABA) receptors. Previous studies in membranes from house fly heads have shown [H-3]EBOB to have high affi nity for the cyclodiene binding site, which was reduced fourfold in cy clodiene-resistant strains. Following our recent identification of sin gle amino acid replacements in the Drosophila GABA receptor gene Rdl c onferring resistance to cyclodienes, we were interested in correlating [H-3]EBOB binding and physiology with specific replacements of alanin e302 in Rdl. Here we report that [H-3]EBOB binding is not detectable i n resistant strains carrying either the resistance-associated alanine3 02> serine (Drosophila melanogaster or D. simulans) or the alanine302> glycine (D. simulans) replacement. Thus, despite high specific bindin g to membranes from susceptible flies, no binding higher than nonspeci fic was observed in resistant preparations. EBOB (100 nM) was also sho wn 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 r eplacement showed 10-fold insensitivity to block. These results indica te that the presence of alanine302 is central to the binding of [H-3]E BOB to GABA receptors containing Rdl subunits and confirm the usefulne ss of this radioligand for the study of this important binding site. ( C) 1995 Academic Press, Inc.