Touroutine Denis, Fox Rebecca M, Von Stetina Stephen E, Burdina Anna, Miller David M, Richmond Janet E
Department of Biology, University of Illinois, Chicago, Illinois 60607, USA.
J Biol Chem. 2005 Jul 22;280(29):27013-21. doi: 10.1074/jbc.M502818200. Epub 2005 May 24.
The Caenorhabditis elegans neuromuscular junction (NMJ) contains three pharmacologically distinct ionotropic receptors: gamma-aminobutyric acid receptors, levamisole-sensitive nicotinic receptors, and levamisole-insensitive nicotinic receptors. The subunit compositions of the gamma-aminobutyric acid- and levamisole-sensitive receptors have been elucidated, but the levamisole-insensitive acetylcholine receptor is uncharacterized. To determine which of the approximately 40 putative nicotinic receptor subunit genes in the C. elegans genome encodes the levamisole-resistant receptor, we utilized MAPCeL, a microarray profiling strategy. Of seven nicotinic receptor subunit transcripts found to be enriched in muscle, five encode the levamisole receptor subunits, leaving two candidates for the levamisole-insensitive receptor: acr-8 and acr-16. Electrophysiological analysis of the acr-16 deletion mutant showed that the levamisole-insensitive muscle acetylcholine current was eliminated, whereas deletion of acr-8 had no effect. These data suggest that ACR-16, like its closest vertebrate homolog, the nicotinic receptor alpha7-subunit, may form homomeric receptors in vivo. Genetic ablation of both the levamisole-sensitive receptor and acr-16 abolished all cholinergic synaptic currents at the NMJ and severely impaired C. elegans locomotion. Therefore, ACR-16-containing receptors account for all non-levamisole-sensitive nicotinic synaptic signaling at the C. elegans NMJ. The determination of subunit composition for all three C. elegans body wall muscle ionotropic receptors provides a critical foundation for future research at this tractable model synapse.
秀丽隐杆线虫神经肌肉接头(NMJ)包含三种药理学上不同的离子型受体:γ-氨基丁酸受体、左旋咪唑敏感型烟碱样受体和左旋咪唑不敏感型烟碱样受体。γ-氨基丁酸受体和左旋咪唑敏感型受体的亚基组成已得到阐明,但左旋咪唑不敏感型乙酰胆碱受体尚未得到表征。为了确定秀丽隐杆线虫基因组中约40个假定的烟碱样受体亚基基因中哪一个编码对左旋咪唑耐药的受体,我们采用了MAPCeL,一种微阵列分析策略。在发现的七种在肌肉中富集的烟碱样受体亚基转录本中,有五种编码左旋咪唑受体亚基,剩下两种可能是对左旋咪唑不敏感的受体候选基因:acr-8和acr-16。对acr-16缺失突变体的电生理分析表明,对左旋咪唑不敏感的肌肉乙酰胆碱电流消失,而删除acr-8则没有影响。这些数据表明,ACR-16与其最接近的脊椎动物同源物烟碱样受体α7亚基一样,可能在体内形成同源受体。对左旋咪唑敏感型受体和acr-16进行基因消融,消除了NMJ处所有胆碱能突触电流,并严重损害了秀丽隐杆线虫的运动能力。因此,含有ACR-16的受体介导了秀丽隐杆线虫NMJ处所有非左旋咪唑敏感型烟碱样突触信号。确定秀丽隐杆线虫体壁肌肉所有三种离子型受体的亚基组成,为在这个易于处理的模型突触上开展未来研究提供了关键基础。