Pharmacological Diversity of nAChRs in Clade III Nematodes: Brugia malayi

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Project Summary 1. Cholinergic anthelmintics, including levamisole and pyrantel, are used for the control of nematode parasites. We have found that cholinergic anthelmintics are not a homogenous functional class of drugs. These anthelmintics select for different subtypes of acetylcholine- gated ion-channel receptors (nAChRs) in muscle of parasitic nematodes. The different nAChR subtypes are produced by varied combinations of five subunit proteins: each subunit may be produced by a different gene. We will exploit our advances with RNAi, quantitative motility studies, molecular pharmacology and patch-clamp to study the diversity and dynamic nature of nAChR subtypes of Brugia malayi: a parasite that causes lymphatic filariasis. This parasite is an excellent specific and general nematode parasite model. It is tractable to study with techniques that permit functional studies of genes that produce the different subunits of the nAChR subtypes involved in parasite neuromuscular transmission. 2. The classic cholinergic anthelmintic, levamisole, selectively activates muscle L-subytpe nAChRs, producing spastic contraction in parasitic nematodes. In Brugia adults, responses to levamisole decline over an hour, but responses to other cholinergic anthelmintics do not. Why is there this loss of anthelmintic effect (tachyphylaxis) and why is there a difference between cholinergic anthelmintics? Here we will identify the dynamic functions and interactions of different nAChR genes and a mechanistic explanation for anthelmintic tachyphylaxis. 3. Our approach in Aim #1 will be to characterize, molecularly and pharmacologically, the four or more nAChRs subtypes present on Brugia somatic muscle. In Aim #2, we will identify the functions of nAChR subunit genes by using RNAi on Brugia adults to produce different phenotypes and to alter muscle responses to different cholinergic anthelmintics. In Aim #3, we will test the hypothesis that populations of receptor subtypes are dynamic, compensating for the effects of anthelmintic exposure; we will determine how the L-subtype nAChRs behave during tachyphylaxis. 4. The proposal is innovative, using a combination of techniques for the study of functional properties of filarial nAChR ion-channels genes. To our knowledge, we are the only lab that has been able to combine these techniques successfully for the study of nematode parasites. 5. The overall impact, by an innovative combination of techniques, we will discover important new information on: ●the functional properties of filarial nAChR genes sensitive to anthelmintics; ●the dynamic nature of different receptor subtypes and; ●the loss of functional receptors and expression of genes associated with anthelmintic tachyphylaxis