Effects of [H-3]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1 The radiolabelled bicyclic dinitrile, [H-3]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([H-3]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35+/-3 nM) and dieldrin (IC50, 93+/-7 nM), displaced [H-3]-BIDN from rootworm membranes. When tested at 100 mu M, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2.]octane[EBOB], 4-t-butyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-thione (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2.]octane (TBOB) and picrotoxin failed to displace 50% of [H-3]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. 2 Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and alpha and beta endosulphan displace bound [H-3]-BIDN from rootworm membranes by a competitive mechanism. 3 Rat brain membranes were also shown to possess a population of saturable, specific [H-3]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [H-3]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. 4 Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffected. 5 Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl- channels to which a number of insecticidally-active molecules bind.