Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor
Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor
复制标题
DOI：
10.1073/pnas.0806933105
复制
发表时间：
2008-11-25
影响因子：
11.1
通讯作者：
Bessereau, Jean-Louis
中科院分区：
文献类型：
--
作者：
Boulin, Thomas;Gielen, Marc;Bessereau, Jean-Louis
左旋咪唑敏感性乙酰胆碱受体（Levamisole-sensitive acetylcholine receptor，L-AChRs）是线虫神经肌肉接头上的一种配体门控离子通道，介导兴奋性神经传递。它们构成了驱虫治疗的主要药物靶标，因为它们可以被线虫特异性胆碱能激动剂如左旋咪唑激活。在秀丽隐杆线虫对左旋咪唑毒性的抗性中进行的遗传筛选确定了对L-AChRs的生物合成不可或缺的基因。这些基因包括5个编码不同AChR亚基的基因和3个编码参与受体组装和运输的辅助蛋白的基因。尽管广泛的分析L-AChRs在体内，药理学和生物物理特性的这些受体已大大阻碍了一个异源表达系统的情况下。使用非洲爪蟾卵母细胞，我们能够重建功能的L-AChRs共表达的5个不同的受体亚基和3个辅助蛋白。引人注目的是，该系统概括了体内受体表达的遗传要求，因为省略这8个基因中的任何一个都会显着损害L-AChR的表达。我们证明了3个α-和2个非α-亚基组装成相同的受体。药理学分析表明，原型胆碱能激动剂尼古丁不能激活L-AChR，而是作为一种有效的变构抑制剂。这些结果强调了辅助蛋白的作用，有效表达的重组神经递质受体和开放的方式，在体外筛选新的驱虫剂。
Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 alpha- and 2 non-alpha-subunits assemble into the same receptor. Pharmacological analysi's reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.