Neurotoxin interactions with ligand-gated ion channels

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The overall goal of this project is to determine the mechanism and site of neurotoxin action on ligand-gated ion channels. Picrotoxin (PTX) is the prototypical neurotoxin, and inhibits both GABAA and glycine receptors. These proteins are structurally related receptor/CI- channel complexes, and are the predominant inhibitory neurotransmitter receptors in the central nervous system. Despite years of study, the site and mechanism of action of PTX is unknown. We will test the hypothesis that PTX acts deep in the channel of the homomeric glycine and the heteromeric GABAA receptor. Because our preliminary results suggest these toxic agents interact at overlapping but distinct domains, we will define the structural determinants for three neurotoxins: 1) picrotoxin, 2) the insecticide dieldrin, and 3) the widely used CNS convulsant pentylenetetrazole (PTZ). Recent work from our lab indicates these neurotoxins also block the 5HT3 receptor, a cation-selective ligand-gated ion channel. These previously undescribed effects on cation-selective channels may provide additional insight into the central actions of these toxic agents. The specific aims of this proposal are to test the following hypotheses: 1) that channel-lining residues near the cytoplasmic aspect of TM2 are the site of action of picrotoxin; 2) that neurotoxin inhibition is dependent on the physical characteristics of the amino acids in this TM2 domain; 3) that the functional domains for PTX, dieldrin and PTZ are overlapping but distinct; and 4) that neurotoxins block the cation-selective 5HT3 receptor through a domain similar to that of the anion- selective channels. A complete understanding of neurotoxin actions in both insects and vertebrate receptors is critical for further development of new and safer insecticides. In addition, because these agents are believed to interact in the ion channel itself, our analysis will provide fundamental information about the characteristics of channel-lining amino acids that influence ion channel gating and permeation. This information may be relevant to several members of the ligand-gated ion channel superfamily.