Cys-loop ligand-gated chloride channels from Dirofilaria immitis: Biological function and pharmacology

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Dirofilaria immitis is a mosquito-transmitted parasitic nematode that primarily infects domestic and wild dogs and is the causative agent of the debilitating disease called heartworm. For the past two decades the only preventative treatments for heartworm are drugs (also called anthelmintics) from the macrocyclic lactone family which primarily target a type of cys-loop receptor called the glutamate-gated chloride channels. However, there are now reports of resistance of this parasite to macrocyclic lactone drugs in certain regions. Therefore, there is a growing need and opportunity for the development of new anthelmintics. The cys-loop family of ligand-gated chloride channels (LGCCs) remains an attractive target for the development of future anthelmintics. These receptors are from a large family of pentameric (i.e. 5 subunits) ligand-gated ion channels that play an important role in the nervous system of parasitic nematodes. They have been shown to play key roles in several biological processes such as movement, feeding and reproduction. In the D. immitis genome there are about 24 genes that encode subunits from this family and most have yet to be characterized. Moreover, we still do not know the role these receptors play in various biological process in D. immitis such as movement. This proposal will serve as the first comprehensive study of the cys-loop LGCC family from this pathogen. A component of this study will involve an evaluation of transcript levels of receptor genes in microfilaria (larval stage) and adult worms. This will help shape further characterization using RNAi targeting various receptor genes with analysis of phenotype using the Worminator system which quantifies changes in worm movement in response to bioactive molecules. Further characterization of D. immitis cys-loop receptors will be completed through our Xenopus expression system for characterization using 2-electrode voltage clamp electrophysiology. Electrophysiology will provide details on the pharmacological (ie drug) profile of various receptors and will be key in helping to identify the natural ligand for previously unknown receptors. Overall this research will uncover new details on the the role of LGCCs in the biology of D. immitis and possibly identify new promising targets for anthelmintic development.  The project will provide excellent training opportunities for highly-qualified personnel as it will not only enhance the technical/research skills of the trainees but will provide them with a deep understanding of a particular biological system from the molecular to organism level.  In addition, we anticipate that further developments in RNAi for parasitic nematodes provided by this research will contribute immensely to the parasitology community.  Finally, since there is an increased risk of transmission of heartworm in Canada due to climate change, this research will greatly benefit this country's commitment to tackling this pathogen.