Binding and physiology of 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) in cyclodiene-resistant Drosophila
1995
Lee, H.J. | Zhang, H.G. | Jackson, M.B. | Ffrench-Constant, R.H.
4'-Ethynyl-4-n-[2,3-3H2]propylbicycloorthobenzoate ([3H]EBOB) is a novel radioligand for the convulsant binding site of vertebrate and invertebrate gamma-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 nM) 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.
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