Ascariasis and hookworm infections are carried by 1.6 billion people throughout the world and in 2 percent of cases cause loss of life. Anthelmintics, including levamisole and related drugs (pyrantel and oxantel), are used to combat nematode parasites, but the development of resistance is a concern. The long-range objective is to improve and protect human health by protecting the efficacy of anthelmintic drugs by controlling and reversing resistance. The objective of the application is to identify mechanisms that regulate the sensitivity of the response of nematode parasite levamisole receptor channels. Our central hypothesis is that competing processes (phosphorylation-dephosphorylation) modulate levamisole responses, and that modification of the processes can produce a decrease or increase in resistance. We developed this hypothesis on the basis of: 1) analysis showing consensus regulatory phosphorylation sites on levamisole receptors; 2) our published and preliminary data showing reduced patency of levamisole receptor channels in resistant nematodes; 3) strong preliminary data that shows levamisole responses are reduced by inhibition of protein kinases. The rationale for the research is that, once mechanisms for regulating levamisole receptor channels are known, pharmacological approaches can be formulated to prevent or overcome resistance, and to maintain the efficacy of levamisole and related anthelmintics. In most experiments we will use electrophysiological techniques on Ascaris suum to examine the properties of the receptor channel. Muscle-flap preparations with current- and voltage-clamp techniques will be used for screening drug effects. We will use muscle-vesicle preparations and patch-clamp technology to measure effects on gating kinetics of levamisole receptor channels of nematode parasites. We will pursue two specific aims to accomplish our current objective: 1) determine mechanisms by which nematode parasites limit their response (P-open) of receptors, and become resistant to levamisole; 2) determine mechanisms that increase P-open values of receptors, in order to reverse resistance to levamisole. We will test our hypothesis that levamisole resistance can be reversed by increased receptor phosphorylation in different species of resistant nematodes. The research is innovative because few groups carry out parasite electrophysiology and others have not developed nematode parasite muscle-vesicle preparations for patch-clamp recordings from levamisole receptor channels. We expect the research to identify mechanisms that decrease levamisole responses (reduce P-open) so parasites become resistant to levamisole and to demonstrate how this resistance can be reversed. The research is significant because application of the results is expected to lead towards methods that will control and overcome resistance to anthelmintics of the levamisole class.
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