Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
J Neurochem. 2012 Sep;122(6):1137-44. doi: 10.1111/j.1471-4159.2012.07867.x. Epub 2012 Aug 3.
The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in pathophysiology of the auditory system, chronic pain, and breast and lung cancers. Alpha-conotoxins, from the predatory marine snail Conus, are potent nicotinic antagonists, some of which are selective for the α9α10 nAChR. Here, we report a two order of magnitude species difference in the potency of α-conotoxin RgIA for the rat versus human α9α10 nAChR. We investigated the molecular mechanism of this difference. Heterologous expression of the rat α9 with the human α10 subunit in Xenopus oocytes resulted in a receptor that was blocked by RgIA with potency similar to that of the rat α9α10 nAChR. Conversely, expression of the human α9 with that of the rat α10 subunit resulted in a receptor that was blocked by RgIA with potency approaching that of the human α9α10 receptor. Systematic substitution of residues found in the human α9 subunit into the homologous position in the rat α9 subunit revealed that a single point mutation, Thr56 to Ile56, primarily accounts for this species difference. Remarkably, although the α9 nAChR subunit has previously been reported to provide the principal (+) binding face for binding of RgIA, Thr56 is located in the (-) complementary binding face.
α9α10 型烟碱型乙酰胆碱受体 (nAChR) 可能是听觉系统病理生理学、慢性疼痛以及乳腺癌和肺癌的潜在靶点。来自掠食性海洋蜗牛 Conus 的α-芋螺毒素是有效的烟碱型拮抗剂，其中一些对α9α10 nAChR 具有选择性。在这里，我们报告了α-芋螺毒素 RgIA 对大鼠和人类α9α10 nAChR 的效力存在两个数量级的种属差异。我们研究了这种差异的分子机制。在非洲爪蟾卵母细胞中异源表达大鼠α9 和人α10 亚基，得到的受体被 RgIA 阻断，其效力与大鼠α9α10 nAChR 相似。相反，表达人α9 和大鼠α10 亚基的受体被 RgIA 阻断，其效力接近人α9α10 受体。系统地将人α9 亚基中发现的残基替换为大鼠α9 亚基中的同源位置，发现一个单一的点突变，即 Thr56 突变为 Ile56，主要导致了这种种属差异。值得注意的是，尽管先前已经报道 α9 nAChR 亚基提供了 RgIA 结合的主要 (+) 结合面，但 Thr56 位于 (-) 互补结合面。
