The overall goal of this research proposal is to determine the mechanism of action of GABAA receptor-specific insecticides and related neurotoxins. A prominent mechanism by which insecticides exert their toxic effects appears to be through an interaction with voltage-gated or ligand-gated ion channels. Evidence suggests that the effects of many insecticides are due to an interaction with the insect GABA receptor. The insect receptor is relatively homologous with the vertebrate GABAA receptor, and consequently many of these insecticides are also toxic to vertebrates. However, the mechanism through which these insecticides and other neurotoxins block the activity of the GABAA receptor has not been determined. Moreover, studies that have attempted to investigate this question have not been conducted using human-derived receptors. Thus, the patch clamp technique will be used to determine the mechanism of neurotoxic action on recombinant human and rat GABAA receptors expressed in human embryonic kidney cells.
The specific aims of this research proposal are to: i) test the hypothesis that neurotoxins exert their inhibitory effects on the recombinant GABAA receptor by decreasing' single-channel open probability; 2) test the hypothesis that the presence of GABA enhances the association rate of the neurotoxins to their binding site(s) on the recombinant GABAA receptor; 3) test the hypothesis that the kinetic interactions of GABA receptor-related neurotoxins are influenced by subunit configuration of the recombinant GABAA receptor; and 4) test the hypothesis that phosphorylation state of the recombinant GABAA receptor modulates the effects of the neurotoxins. The results of these studies will enhance our understanding of how neurotoxic agents exert their toxic effects on the mammalian central nervous system. This information should be useful in the development of insecticides that are less toxic to humans and other vertebrates. Results from these studies will also provide general information about modulation of ligand receptor interactions for the GABAA receptor; this knowledge may be relevant to the whole superfamily of ligand-gated ion channels.
