HOUSE-FLY HEAD GABA-GATED CHLORIDE CHANNEL - [H-3] ALPHA-ENDOSULFAN BINDING IN RELATION TO POLYCHLOROCYCLOALKANE INSECTICIDE ACTION

This study attempts to use [H-3]alpha-endosulfan to examine directly t he binding site(s) for cyclodienes, lindane and toxaphene (collectivel y referred to as the polychlorocycloalkane or PCCA insecticides) in th e 4-aminobutyric acid (GABA)-gated chloride channel. [H-3]alpha-Endosu lfan was prepared by reduction of hexachloronorbornenedicarboxylic anh ydride with sodium borotritide, then coupling the labelled alcohol wit h thionyl chloride followed by HPLC purification (35 Ci mmol-1, >99% r adiochemical purity). This new candidate radioligand readily partition s into lipid membranes and undergoes indiscriminate adsorption to surf aces resulting in high levels of non-specific binding. This makes it v ery difficult to differentiate the small portion of specific binding a t the site relevant to toxic action. This problem was partially circum vented by incubating [H-3]alpha-endosulfan (0.1 nM) with house-fly hea d membranes (0.2 mg protein) for 70 min at 22-degrees-C giving 23 (+/- 4)% specific binding (40 fmol mg-1 protein) determined as the differen ce between the radioligand alone and on preincubation for 15 min with unlabelled alpha-endosulfan (final concentration 100 nM). This procedu re is not appropriate for determination of saturation isotherms and st andard binding kinetics. However, the effectiveness of 16 PCCAs (also at 100 nM final concentration) in blocking the specific binding of [H- 3]alpha-endosulfan is generally consistent with their relative potenci es as inhibitors of (4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2]octane ([H-3]EBOB) binding suggesting that the binding site for both [H-3]al pha-endosulfan and the PCCAs is part of the GABA-gated chloride channe l. Insecticidal channel blockers of other types (e.g. picrotoxinin, tr ioxabicyclooctanes, a dithiane, and phenylpyrazoles) and GABA are poor inhibitors of [H-3]alpha-endosulfan binding relative to their potenci es as inhibitors of [H-3]EBOB binding. It therefore appears that the P CCAs compete directly for the [H-3]alpha-endosulfan site, whereas the other channel blockers bind with different inhibition kinetics or at a site more closely coupled to the EBOB than the alpha-endosulfan bindi ng domain.