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

双丝虫是一种由蚊子传播的寄生线虫，主要感染家犬和野狗，是一种名为心虫的衰弱疾病的病原体。在过去的二十年里，对心虫的唯一预防性治疗是来自大环内酯家族的药物(也称为驱虫药)，这些药物主要针对一种名为谷氨酸门控氯离子通道的环状受体。然而，现在有报道称，在某些地区这种寄生虫对大环内酯类药物具有抗药性。因此，开发新的驱虫药的需求和机遇越来越大。配体门控氯通道(LGCCs)环家族仍然是未来驱虫药开发的一个有吸引力的靶标。这些受体来自一个五聚体(即5个亚基)配基门控离子通道大家族，它们在寄生线虫的神经系统中发挥着重要作用。它们已被证明在几个生物过程中发挥关键作用，如运动、摄食和繁殖。在蠕形虫基因组中，大约有24个基因编码这个家族的亚基，其中大多数还没有确定。此外，我们还不知道这些受体在蠕形虫运动等各种生物学过程中所起的作用。这项建议将成为对这种病原体的Cys-loop LGCC家族的第一次全面研究。这项研究的一个组成部分将包括评估微丝虫(幼虫期)和成虫中受体基因的转录水平。这将有助于通过针对不同受体基因的RNAi来形成进一步的特征，并使用蠕虫系统来分析表型，该系统可以量化蠕虫运动对生物活性分子的响应变化。通过我们的非洲爪哇表达系统，我们将利用双电极电压钳电生理学完成对金线虫Cys-loop受体的进一步鉴定。电生理学将提供各种受体的药理(即药物)特征的细节，并将是帮助识别先前未知受体的天然配体的关键。总体而言，这项研究将揭示LGCCs在寄生线虫生物学中的作用的新细节，并可能为驱虫药的开发找到新的有前途的靶点。该项目将为高素质的人员提供极好的培训机会，因为它不仅将提高受训者的技术/研究技能，还将使他们从分子到生物水平对特定生物系统有深入的了解。此外，我们预计，通过本研究提供的针对寄生线虫的RNAi的进一步发展将对寄生虫界做出巨大贡献。最后，由于气候变化增加了加拿大心线虫的传播风险，这项研究将极大地有利于该国致力于解决这种病原体。

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.