Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues.
Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues.
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DOI：
10.1371/journal.pone.0151183
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Ulens C
中科院分区：
文献类型：
--
作者：
Wijckmans E;Nys M;Debaveye S;Brams M;Pardon E;Willegems K;Bertrand D;Steyaert J;Efremov R;Ulens C
半胱氨酸环受体是跨膜配体门控离子通道，参与快速兴奋性和抑制性神经传递。通过X射线晶体学或电子显微镜测定的这些离子通道的三维结构揭示了关于配体识别、通道门控和离子电导的分子机制的有价值的信息。为了扩展和验证目前的见解，我们在这里提出了有前途的候选人进行进一步的结构研究。我们报告的生化和功能特性的半胱氨酸环受体同源物中确定的蛋白质组的Alvinella pompejana，嗜极端，多毛类环节动物发现在热液喷口底部的太平洋。选择了7个同源物，命名为Alpo 1 -7。其中5个，Alpo 2 -6，在本研究之前尚未鉴定。双电极电压钳实验表明，野生型Alpo 5和Alpo 6与人甘氨酸受体α亚基具有高度的序列同源性，是一种能被甘氨酸、GABA和牛磺酸激活的阴离子选择性通道。此外，在昆虫细胞中表达后，荧光尺寸排阻色谱实验表明，四个同源物，Alpo 1，Alpo 4，Alpo 6和Alpo 7，可以通过各种洗涤剂从膜中提取出来，同时保持其低聚状态。最后，Alpo 1、Alpo 4和Alpo 6的大规模纯化工作产生了毫克量的生物化学稳定的单分散蛋白。总之，我们的研究结果建立了环节动物中甘氨酸受体的进化保守性，并为未来的结构研究铺平了道路。
Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies.