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

犬恶丝虫（Dirofilaria immitis）是一种蚊子传播的寄生线虫，主要感染家犬和野狗，是一种名为心丝虫的衰弱性疾病的病原体。在过去的二十年中，心丝虫的唯一预防性治疗是来自大环内酯家族的药物（也称为驱虫剂），其主要靶向一种称为谷氨酸门控氯离子通道的cys环受体。然而，现在有报告说，这种寄生虫在某些地区对大环内酯类药物产生耐药性。因此，开发新的驱虫剂的需求和机会越来越大。配体门控氯离子通道（LGCCs）的cys环家族仍然是未来驱虫药开发的一个有吸引力的目标。这些受体来自五聚体（即5个亚基）配体门控离子通道的大家族，其在寄生线虫的神经系统中起重要作用。它们已被证明在几个生物过程中发挥关键作用，如运动，进食和繁殖。在地方犬基因组大约有24个基因编码来自该家族亚基，且大多数尚未被表征。此外，这些受体在D.免疫系统如运动。这一提议将作为该病原体的cys环LGCC家族的第一个全面研究。这项研究的一个组成部分将涉及微丝蚴（幼虫阶段）和成虫蠕虫基因的转录水平的评价。这将有助于使用靶向各种受体基因的RNAi进行进一步表征，并使用Worminator系统进行表型分析，该系统定量响应生物活性分子的蠕虫运动变化。对D.通过我们的爪蟾表达系统，使用2电极电压钳电生理学进行表征，将完成犬Cys环受体的表达。电生理学将提供各种受体的药理学（即药物）特征的详细信息，并将是帮助识别先前未知受体的天然配体的关键。总的来说，这项研究将揭示LGCC在D. immitis，并可能确定新的有希望的目标驱虫剂的发展。 该项目将为高素质人员提供极好的培训机会，因为它不仅将提高受训人员的技术/研究技能，而且将使他们从分子到生物体一级深入了解特定的生物系统。 此外，我们预计这项研究提供的寄生线虫RNAi的进一步发展将为寄生虫学社区做出巨大贡献。 最后，由于气候变化，加拿大的心丝虫传播风险增加，这项研究将大大有利于该国应对这种病原体的承诺。

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.