Baylis H A, Matsuda K, Squire M D, Fleming J T, Harvey R J, Darlison M G, Barnard E A, Sattelle D B
Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK.
Recept Channels. 1997;5(3-4):149-58.
The molecular cloning and functional co-expression of a novel nicotinic acetylcholine receptor (nAChR) non-alpha subunit gene, acr-3, is described. Previously we determined the sequence and demonstrated the functional co-expression of acr-2, a nAChR non-alpha subunit gene from Caenorhabditis elegans. Analysis of the acr-2 genomic DNA revealed the existence of another potential nAChR subunit gene, acr-3, in the same orientation, only 281 bp downstream of acr-2. A cDNA containing the entire acr-3 coding sequence was isolated by RT-PCR and sequenced. The predicted protein contains the conserved features typical of nAChR non-alpha subunits and most closely resembles other invertebrate nAChR non-alpha polypeptides. Unusually, the highly conserved glycine residue (equivalent to residue 240 in the Torpedo alpha subunit) upstream of transmembrane domain 2 (m2) is replaced by a serine residue in ACR-3. When acr-3 cDNA was injected alone into Xenopus oocytes no levamisole-gated channel activity was observed. However when co-expressed with a C. elegans alpha subunit (UNC-38), ACR-3 contributed to the formation of levamisole-gated channels. The response of this hetero-oligomer to levamisole (100 microM) was reduced by the nAChR antagonists mecamylamine (1 microM) and d-tubocurarine (10 microM).
本文描述了一种新型烟碱型乙酰胆碱受体（nAChR）非α亚基基因acr-3的分子克隆及功能共表达。此前我们已确定了秀丽隐杆线虫nAChR非α亚基基因acr-2的序列，并证明了其功能共表达。对acr-2基因组DNA的分析揭示，在acr-2下游仅281 bp处，以相同方向存在另一个潜在的nAChR亚基基因acr-3。通过逆转录聚合酶链反应（RT-PCR）分离出包含完整acr-3编码序列的cDNA并进行了测序。预测的蛋白质含有nAChR非α亚基典型的保守特征，与其他无脊椎动物nAChR非α多肽最为相似。不同寻常的是，跨膜结构域2（m2）上游高度保守的甘氨酸残基（等同于电鳐α亚基中的第240位残基）在ACR-3中被丝氨酸残基取代。当单独将acr-3 cDNA注射到非洲爪蟾卵母细胞中时，未观察到左旋咪唑门控通道活性。然而，当与秀丽隐杆线虫α亚基（UNC-38）共表达时，ACR-3有助于左旋咪唑门控通道的形成。烟碱型乙酰胆碱受体拮抗剂美加明（1 μM）和d-筒箭毒碱（10 μM）可降低这种异源寡聚体对左旋咪唑（100 μM）的反应。
