The long-term goal of the proposed study is to elucidate the mechanism by which neuroactive insecticides exert their toxic actions on mammals. Specifically, the interactions of the insecticides with ion channels will be studied by using patch clamp techniques as applied to mammalian neurons isolated from the dorsal root ganglion, cerebellum, and hippocampus of the rat. The major target sites of pyrethroids and dieldrin/lindane are the voltage-gated sodium channel and the GABA-A receptor-channel, respectively. Several important breakthroughs accomplished recently have raised new critical questions regarding the mechanisms by which these insecticides modulate the respective target site. Type II (alpha-cyano) pyrethroids act on the sodium channel in a manner somewhat different from type I (no alpha-cyano) pyrethroids. Differential actions of type II pyrethroids on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels will be analyzed with respect to the percentages of the modified sodium channels (project Aa). Negative temperature dependence of pyrethroids is a critical factor for the selective toxicity between mammals and invertebrates, and the mechanisms will be elucidated in terms of single sodium channel kinetics for both type I and type II pyrethroids (projects Ab and Ac). The discovery of the selective vitamin E block of the sodium channel modified by type I pyrethroids has opened the door for development of antidotes, and the underlying mechanisms for both types of pyrethroids will be explored with special emphasis on the open channel block by vitamin E (project Ad). The newly discovered dual action (stimulation and suppression) of dieldrin and lindane on the GABA receptor channel has raised important questions. Channel state dependence of action will be studied by analyzing use-dependent onset and recovery (project Ba). The dual effect is likely to due to the direct stimulating action and desensitization. The mechanism of desensitization caused by dieldrin and lindane will be studied by analyzing the drug dissociation rate from the binding site and single-channel kinetics (project Bb). The roles of GABA receptor subunits, especially the alpha1, alpha6, gamma2 and delta subunits, in the dual action of dieldrin and lindane will be determined by using the human embryonic cell line expressing various subunits with due consideration of subconductance states of single channels (project Bc). The GABA receptor subunits undergo developmental changes, and therefore, the actions of dieldrin and lindane are expected to change. This hypothesis will be studied during in vitro cell development and in vivo animal development (project Bd). The proposed study will provide useful and critical information for development of newer and safer insecticides and of prophylactic and treatment methods for insecticide intoxication.
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