David M. Soderlund - US grants

The proposed research will test the hypothesis that pyrethroid insecticides exert their neurotoxic action by binding to an unique and as-yet-uncharacterized stereospecific receptor site associated with nerve membrane sodium channels. Two complementary approaches will be used: a stereospecific receptor binding assay using optically pure pyrethroid radiligands; and an assay of pyrethroid stimulation of veratridinedependent radiosodium flux through nerve synaptosomal sodium channels. Data will be obtained from these two approaches using a series of pyrethroids exemplifying critical structure-activity relationships in synaptosomal preparations from mouse brain and cockroach nerve cord. Comparison of these data with in vivo measurements of intrinsic toxicity in both mice and cockroaches will (1) allow critical evaluation of the stereospecific binding site as a toxicologically significant pyrethroid receptor, (2) determine the relationship between stereospecific binding and pyrethroid-dependent potentiation of sodium channel activation, and (3) define intrinsic differences in the pyrethroid receptor of target and non-target species. Subsequent studies will explore the binding and pharmacological effects of pyrethroids on purified and reconstituted sodium channel preparations from mouse brain. Results of these studies will contribute to the resolution of the current controversy over the mode of action of these important insecticides. They will also provide experimental systems appropriate for identifying specific antidotes for pyrethroid intoxication in mammals and for developing intrinsically selective new insecticides by exploiting receptor differences between mammals and insects.

The proposed research will test the hypothesis that pyrethroid insecticides exert their neurotoxic action by binding to an unique and as-yet-uncharacterized stereospecific receptor site associated with nerve membrane sodium channels. Two complementary approaches will be used: a stereospecific receptor binding assay using optically pure pyrethroid radiligands; and an assay of pyrethroid stimulation of veratridinedependent radiosodium flux through nerve synaptosomal sodium channels. Data will be obtained from these two approaches using a series of pyrethroids exemplifying critical structure-activity relationships in synaptosomal preparations from mouse brain and cockroach nerve cord. Comparison of these data with in vivo measurements of intrinsic toxicity in both mice and cockroaches will (1) allow critical evaluation of the stereospecific binding site as a toxicologically significant pyrethroid receptor, (2) determine the relationship between stereospecific binding and pyrethroid-dependent potentiation of sodium channel activation, and (3) define intrinsic differences in the pyrethroid receptor of target and non-target species. Subsequent studies will explore the binding and pharmacological effects of pyrethroids on purified and reconstituted sodium channel preparations from mouse brain. Results of these studies will contribute to the resolution of the current controversy over the mode of action of these important insecticides. They will also provide experimental systems appropriate for identifying specific antidotes for pyrethroid intoxication in mammals and for developing intrinsically selective new insecticides by exploiting receptor differences between mammals and insects.

DESCRIPTION: (Adapted from the Investigator's Abstract) Synthetic pyrethroids are an important class of neurotoxic insecticides used worldwide in agriculture and public health. Pyrethroids cause toxic effects in vertebrate nerves and muscles by modifying the normal function of voltage-sensitive sodium channels. Sodium channels exist in multiple isoforms that exhibit differential tissue distribution and developmental regulation and are encoded by members of a multigene family. Previous studies of pyrethroid action have not considered the differential sensitivity of sodium channel isoforms as a determining factor in the toxicity of these compounds. The overall objectives of the proposed research are to test the hypotheses that vertebrate sodium channel isoforms exhibit differential sensitivity to synthetic pyrethroids and that these differences in sensitivity are important determinants of the nature and severity of toxicological responses to pyrethroid exposure. Specific experimental goals are: (1) to define the actions of pyrethroid insecticides on cloned rat sodium channel isoforms expressed in Xenopus oocytes; (2) to correlate the effects of pyrethroids on sodium channel isoforms with acute and chronic neurotoxic effects; (3) to compare the actions of pyrethroids on homologous rat and human sodium channel isoforms expressed in oocytes; and (4) to map the structural determinants of differential pyrethroid sensitivity among sodium channel isoforms by the construction, heterologous functional expression and pharmacological characterization of chimeric and specifically mutated sodium channels. Cloned sodium channel isoforms will be expressed in Xenopus oocytes and the actions of pyrethroids on the channels will be assessed under voltage clamp. Results of these studies will define the role of different sodium channel isoforms in pyrethroid toxicity. Comparisons of rat and human sodium channel isoforms will determine whether rat isoforms are valid models for the sensitivity of homologous human isoforms to pyrethroids. Studies with chimeric and mutated isoforms will identify channel domains that determine pyrethroid sensitivity and will contribute to knowledge of structure-function relationships for sodium channels.

[unreadable] DESCRIPTION (provided by applicant): Pyrazoline-type insecticides (PTIs) are a new class of insecticides exemplified by indoxacarb, the first compound of the class registered for commercial use in the United States. PTIs exert their neurotoxic effects in insects by selectively blocking sodium channels in depolarized cells. PTIs also selectively block mammalian sodium channels at membrane potentials that promote slow sodium channel inactivation. This effect is similar in many respects to the state-dependent sodium channel block caused by several classes of therapeutic drugs (e.g., local anesthetics, antiarrhythmics, anticonvulsants, neuroprotectants and analgesics) that bind to the "local anesthetic receptor" site of sodium channels. As a result, the local anesthetic receptor has been implicated indirectly as the site of PTI action. The proposed research will test the hypothesis that PTIs block sodium channels by binding to the local anesthetic receptor site of slow-inactivated sodium channels. Specific experimental aims for the proposed funding period are: (1) to define the actions of PTIs on cloned rat sodium channel isoforms and subunit combinations expressed in Xenopus oocytes; (2) to elucidate the mechanism of voltage-dependent sodium channel block by PTIs; and (3) to map the structural determinants of PTI binding to sodium channels by site-directed mutagenesis. Cloned sodium channel isoforms will be expressed in Xenopus oocytes and the actions of PTIs on expressed channels will be assessed using electrophysiological recordings of sodium currents under voltage-clamp conditions. Results of these studies will define the differential sensitivity of sodium channel isoforms to PTIs, elucidate the mechanism of state-dependent sodium channel block by these compounds, and identify the molecular determinants of PTI binding to sodium channels. This research will provide new insight into the structure of the local anesthetic receptor and its role in the neurotoxic actions of PTIs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Synthetic pyrethroids, an important class of neurotoxic insecticides used worldwide in agriculture and public health, cause toxic effects in animals by modifying the normal function of voltage-sensitive sodium channels. Sodium channels in mammalian tissues exist as multiple isoforms that exhibit differential tissue distribution and developmental regulation and are encoded by members of a multigene family. Previous studies of pyrethroid action have not considered the differential sensitivity of sodium channel isoforms as a determining factor in the toxicity of these compounds. The proposed research will test the hypothesis that differences in the pyrethroid sensitivity of mammalian sodium channel isoforms are important determinants of the nature and severity of pyrethroid intoxication. Specific experimental aims are: (1) to define the actions of pyrethroid insecticides on cloned rat sodium channel isoforms expressed in frog (Xenopus laevis) oocytes or transfected mammalian cells;(2) to compare the actions of pyrethroids on orthologous rat and human sodium channel isoforms expressed in Xenopus oocytes or trnasfected mammalian cells;and (3) to map the the structural determinants of pyrethroid action by the construction, functional expression and pharmacological characterization of specifically mutated sodium channels. Cloned sodium channel isoforms will be expressed in Xenopus oocytes or transiently transfected mammalian cells (e.g., HEK-293) and the actions of pyrethroid insecticides on expressed channels will be assessed using electrophysiological recordings of macroscopic sodium currents under two-electrode voltage-clamp or whole cell patch-clamp conditions. Structural determinants of pyrethroid sensitivity will be identified by site-directed mutagenesis, expression, and pharmacological comparison of native a mutated sodium channel isoforms. Results of these studies will define the role of different sodium channel isoforms in pyrethroid toxicity, establish whether rat sodium channels are valid toxicological models for the sensitivity of human sodium channels to pyrethroids, and identify sodium channel domains that determine pyrethroid sensitivity.