Functional characterization of a novel family of acetylcholine-gated chloride channels in Schistosoma mansoni.
Functional characterization of a novel family of acetylcholine-gated chloride channels in Schistosoma mansoni.
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DOI：
10.1371/journal.ppat.1004181
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发表时间：
2014-06
期刊：
影响因子：
6.7
通讯作者：
Ribeiro P
中科院分区：
文献类型：
--
作者：
MacDonald K;Buxton S;Kimber MJ;Day TA;Robertson AP;Ribeiro P
乙酰胆碱是哺乳动物神经肌肉系统的典型兴奋性神经递质。然而，在吸虫寄生虫曼氏血吸虫中，胆碱能刺激导致肌肉松弛和弛缓性麻痹，这表明了一种抑制性作用模式。缺乏关于这种抑制作用的药理学机制的信息。在这里，我们使用了一种技术的组合，以评估胆碱能受体的作用，海马运动功能。乙酰胆碱的神经肌肉作用通常由烟碱受体（nAChR）家族的门控阳离子通道介导。生物信息学分析鉴定了S. mansoni基因组，但有趣的是，这些亚基中几乎有一半携带通常与氯化物选择性相关的基序。这些假定的线粒体乙酰胆碱门控氯离子通道（SmACCs）在进化上与线虫不同，并在nAChR的大家族中形成了一个独特的分支。药理学和RNA干扰（RNAi）的行为屏幕被用来评估的SmACCs在幼虫运动功能的作用。用拮抗剂治疗产生与SmACC的RNAi抑制相同的效果;两者都导致与抑制性神经肌肉介质的废除一致的高运动表型。然后针对两种SmACC产生抗体用于免疫定位研究。SmACC-1和SmACC-2定位于支配体壁肌肉的周围神经系统区域，但两者似乎都不直接在肌肉组织上表达。一个基因SmACC-1在HEK-293细胞中表达，并使用碘通量测定进行表征。结果表明，SmACC-1形成了一个功能性的同源氯离子通道，并被一组胆碱能激动剂选择性激活。在这项研究中描述的结果确定了一个新的分支的烟碱氯离子通道，作为抑制性调制器的突触体神经肌肉功能。此外，用于表征SmACC-1的碘化物通量测定代表了针对这些独特的寄生虫离子通道进行药物筛选的新的高通量工具。血吸虫病是一种广泛的慢性疾病，影响发展中国家2亿多人。目前没有疫苗，治疗依赖于使用一种药物吡喹酮。关于吡喹酮疗效降低以及其对童虫幼虫无效的报告强调了开发新疗法的必要性。由于其在各种基本生物过程中的重要性，干扰溶酶体运动功能提供了有希望的治疗靶点。胆碱能系统以前已被证明是寄生虫运动的主要调节剂。在这项研究中，我们已经描述了一种新的进化枝的乙酰胆碱门控氯离子通道（SmACCs），作为这一途径的抑制性调节剂。我们的研究结果表明，这些受体在人类宿主中不存在，并间接调节抑制性神经肌肉反应，使其成为一个有吸引力的药物靶点。我们还验证了一种新的功能检测来表征这些受体，这可能是修改为未来使用的寄生虫氯离子通道的高通量药物筛选方法。
Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the iodide flux assay used to characterize SmACC-1 represents a new high-throughput tool for drug screening against these unique parasite ion channels. Schistosomiasis is a widespread, chronic disease affecting over 200 million people in developing countries. Currently, there is no vaccine available and treatment depends on the use of a single drug, praziquantel. Reports of reduced praziquantel efficacy, as well as its ineffectiveness against larval schistosomula highlight the need to develop new therapeutics. Interference with schistosome motor function provides a promising therapeutic target due to its importance in a variety of essential biological processes. The cholinergic system has been shown previously to be a major modulator of parasite motility. In this study, we have described a novel clade of schistosome acetylcholine-gated chloride channels (SmACCs) that act as inhibitory modulators of this pathway. Our results suggest that these receptors are absent in the human host and indirectly modulate inhibitory neuromuscular responses, making them an attractive drug-target. We have also validated a new functional assay to characterize these receptors, which may be modified for future use as a high-throughput drug screening method for parasite chloride channels.
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影响因子：
1.5
作者：
Boyle, JP;Wu, XJ;Yoshino, TP
通讯作者：
Yoshino, TP
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作者：
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哺乳动物烟碱乙酰胆碱受体：从结构到功能。
影响因子：
33.6
作者：
Albuquerque EX;Pereira EF;Alkondon M;Rogers SW
通讯作者：
Rogers SW
影响因子：
2.1
作者：
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通讯作者：
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影响因子：
3.8
作者：
El-Shehabi F;Taman A;Moali LS;El-Sakkary N;Ribeiro P
通讯作者：
Ribeiro P