EFFECTS OF [H-3] BIDN, A NOVEL BICYCLIC DINITRILE RADIOLIGAND FOR GABA-GATED CHLORIDE CHANNELS OF INSECTS AND VERTEBRATES

1 The radiolabelled bicyclic dinitrile, oromethyl-bicyclo[2.2.1]heptan e-2,2-dicarbonitrile ([H-3]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undec impunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachl or (IC50, 35+/-3 nM) and dieldrin (IC50, 93+/-7 nM), displaced [H-3]-B IDN from rootworm membranes. When tested at 100 mu M, -n-propyl-2,6,7- trioxabicyclo[2.2.2.]octane[EBOB], ,6,7-trioxa-1-phosphabicyclo[2.2.2] octane-1-thione (TBPS), phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2.]oc tane (TBOB) and picrotoxin failed to displace 50% of [H-3]-BIDN bindin g to rootworm membranes indicating that the bicyclic dinitrile radioli gand 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 satur able, specific [H-3]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the inse cticidal 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 blo cked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffe cted. 5 Expression in Xenopus oocytes of either rat brain mRNA, or cDN A-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in f unctional, homo-oligomeric GABA receptors that were blocked by BIDN. T hus, BIDN probes a novel site on GABA-gated Cl- channels to which a nu mber of insecticidally-active molecules bind.