Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor
Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor
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DOI：
10.1093/molbev/msu258
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发表时间：
2014-12-01
影响因子：
10.7
通讯作者：
Belen Elgoyhen, Ana
中科院分区：
文献类型：
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作者：
Lipovsek, Marcela;Fierro, Angelica;Belen Elgoyhen, Ana
烟碱型乙酰胆碱受体是一个配体门控的非选择性阳离子通道家族，参与中枢和外周神经系统的基本生理过程。钙通过配体门控离子通道进入的程度定义了它们的不同功能。在耳蜗毛细胞中表达的α 9 α 10烟碱胆碱能受体是该家族的一个特殊成员，因为它显示出跨物种钙渗透性程度的差异。特别是，哺乳动物α 9 α 10受体是表现出最高钙选择性的配体门控离子通道。这种获得的差异性质提供了研究蛋白质功能如何形成沿着进化历史的独特机会，通过跟踪其进化记录和实验定义所涉及的氨基酸变化。我们已经应用了祖先序列重建的分子进化方法，结合分子动力学模拟和基于进化的诱变策略，以追踪产生高钙渗透性烟碱α 9 α 10哺乳动物受体的分子事件。在α 9亚基中只有三个特定的氨基酸取代直接参与。这些位于细胞外前庭和通道孔的出口处，而不是如先前所认为的位于蛋白质的跨膜区2处。此外，我们发现，这三个关键的取代只增加钙渗透性的上下文中的哺乳动物，但不是鸟类受体，强调整体蛋白质结构的相关性定义的功能特性。这些结果强调了跟踪蛋白质序列进化获得的变化的重要性，这些变化是配体门控离子通道的基本功能特性的基础。
Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The alpha 9 alpha 10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian alpha 9 alpha 10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic alpha 9 alpha 10 mammalian receptor. Only three specific amino acid substitutions in the alpha 9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.