GABA-gated chloride channel: Binding site for 4′-ethynyl-4- n-[2,3- 3H 2]propylbicycloorthobenzoate ([ 3H]EBOB) in vertebrate brain and insect head

Abstract

4′-Ethynyl-4- n-[2,3- 3H 2]propylbicycloorthobenzoate ([ 3H]EBOB) is an improved radioligand for the noncompetitive blocker site of the vertebrate and fly γ-aminobutyric acid (GABA)-gated chloride channel. It binds with high affinity ( K d 1–4 n M) to a single, saturable site in the GABA-gated chloride channel of human, dog, mouse, and chicken brain assayed at 37°C, of fish brain at 12°C, and of adult house fly and fruit fly heads at 22°C with specific binding of 51–93%. The B max's (pmol/mg protein) are 0.9 – 5.7 for the vertebrates and 0.2 – 0.4 for the insects. Association and dissociation rates fall in the ranges of 0.012 to 0.063 min −1 n M −1 for k +1 and 0.006 to 0.046 min −1 for k −1. The vertebrate preparations are much more sensitive than the insect preparations to GABA and two GABA mimetics (3-aminopropanesulfonic acid and muscimol). Considerable specificity is evident between vertebrates and insects in the inhibitory potencies of various insecticides with the following average selectivity ratios (IC 50 vertebrates/IC 50 flies): t-butylbicyclophosphorothionate and 4- t-butyl-1-ethynyl-2,6,7-trioxabicyclo[2.2.2]octane, 0.04 – 0.18; α-endosulfan, 12-ketoendrin, toxaphene, picrotoxinin, and 2 eq-(4-bromophenyl)-5- t-butyl-1,3-dithiane-2,2-dioxide, 3–8; lindane, 130; and avermectin B 1a and moxidectin, 615–>714. It therefore appears that receptor-site specificity may contribute to species selectivity.