Glenn H. Dillon - US grants

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.

DESCRIPTION (provided by applicant): Carisoprodol (Soma(R)) is a centrally-acting skeletal muscle relaxant frequently prescribed for the treatment of acute musculoskeletal conditions. In recent years, there has been increasing concern regarding carisoprodol's potential as a drug of abuse. In 1991, carisoprodol was ranked 54th among 234 drugs with abuse potential. Only eight years later, the Drug Abuse Warning Network identified carisoprodol as the 14th most abused drug, ranking higher than oxycodone, methadone, and LSD. In support of this, recent reports have substantiated the dangers involved in carisoprodol abuse, including severe withdrawal leading to seizures and death. However, its mechanism of action remains unclear. Until recently, it was widely accepted that the sedative effects of carisoprodol were predominantly due to its metabolite, meprobamate. However, preliminary in vivo and in vitro studies conducted in our laboratory demonstrate that carisoprodol itself is active, and its actions appear to be mediated via novel modulation of the GABAA receptor (GABAAR), the predominant inhibitory neurotransmitter receptor in mammalian brain. This may underlie the capacity of carisoprodol to enhance the sedative effects of CNS depressants, contributing to its potential for abuse. Indeed, concerns about the rise in abuse of carisoprodol have led the Drug Enforcement Agency to contact the National Institute on Drug Abuse for assistance in determining the abuse liability of carisoprodol. Based on our preliminary findings, we hypothesize that carisoprodol itself causes depressive CNS effects, and that these actions are mediated via a novel interaction with the GABAA receptor. The overall goal of the proposed study is to identify the mechanisms that mediate carisoprodol abuse, at both the behavioral and molecular level. We will utilize both in vivo (drug discrimination and observation of intoxication, tolerance and withdrawal, and pharmacokinetic analysis) and in vitro (assessment of carisoprodol on specific configurations of recombinant GABAA receptors) approaches to address the following specific aims: 1) To characterize the discriminative stimulus effects of carisoprodol;2) To investigate whether carisoprodol produces effects on its own in vivo or requires being metabolized to meprobamate;3) To assess the extent of carisoprodol dependence and severity of withdrawal;4) To identify GABAA receptor subunits critical for allosteric modulatory and direct gating effects of carisoprodol;and 5) To identify GABAA receptor subunit domains that confer carisoprodol sensitivity. Given the present and potential dangers posed by carisoprodol abuse, it is of crucial importance to determine the mechanism of action of this drug. Our proposed studies will substantially increase our understanding of the mechanism of action of carisoprodol as a therapeutic agent and as a drug of abuse. This knowledge may provide insight into treating carisoprodol dependence, and withdrawal. PUBLIC HEALTH RELEVANCE: The muscle relaxant Carisoprodol (Soma(R)) is being abused increasingly at an alarming rate;withdrawal from overuse of this drug can cause seizures and even death. There is an urgent need to develop new drugs that can treat these symptoms, as well as new drugs that are less likely to be abused. The proposed studies will provide the fundamental information needed to address both of these current unmet medical needs.