Pharmacological Diversity of nAChRs in Clade III Nematodes: Brugia malayi

项目摘要 1.胆碱能驱虫药，包括左旋咪唑和噻嘧啶，用于控制 线虫寄生虫我们发现胆碱能驱虫药不是同质的， 药物的功能类别。这些驱虫药选择不同亚型的乙酰胆碱- 寄生线虫肌肉中的门控离子通道受体（nAChRs）。不同 nAChR亚型由五种亚基蛋白的不同组合产生：每个亚基 可能是由不同的基因产生的我们将利用我们在RNAi、定量 动力学研究、分子药理学和膜片钳研究的多样性和动态性 马来丝虫nAChR亚型的性质：引起淋巴丝虫病的寄生虫。这 寄生虫是一个很好的特异性和一般性线虫寄生虫模型。研究起来容易 利用允许对产生不同亚基的基因进行功能研究的技术， 参与寄生虫神经肌肉传递的nAChR亚型。 2.经典的胆碱能驱虫药左旋咪唑，选择性激活肌肉L-亚型， nAChR，在寄生线虫中产生痉挛性收缩。在布鲁日成人中， 左旋咪唑在一小时内下降，但对其他胆碱能驱虫剂的反应不会。 为什么会有这种驱虫效果的丧失（快速耐受），为什么会有差异 胆碱能驱虫药之间的区别吗在这里，我们将确定动态函数， 不同nAChR基因的相互作用和驱虫药的机制解释 快速耐受 3.我们在目标#1中的方法将是从分子和生物学上表征这四种 或更多nAChR亚型存在于丝虫属躯体肌肉上。在目标#2中，我们将确定 nAChR亚基基因的功能，通过使用RNAi在Brugia成虫上产生不同的 表型和改变肌肉对不同胆碱能驱虫剂的反应。在目标3中， 我们将检验这样一个假设，即受体亚型的群体是动态的， 对于驱虫剂暴露的影响，我们将确定L-亚型nAChRs 在快速耐受期间表现。 4.该建议具有创新性，采用了功能性相结合的技术进行研究 丝虫nAChR离子通道基因的特性。据我们所知，我们是唯一一个 已经能够成功地将这些技术联合收割机用于线虫寄生虫的研究。 5.整体的影响，通过创新的技术组合，我们将发现重要的 新的信息：●丝虫nAChR基因的功能特性敏感， 驱虫药; ●不同受体亚型的动态性质; ●功能丧失 与驱虫药快速耐受相关的受体和基因表达

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