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Functional and biochemical characterization of Alvinella pompejana cys-loop receptor homologues
Wijckmans, E.; Nys, M.; Debaveye, S.; Brams, M.; Pardon, E.; Willegems, K.; Bertrand, D.; Steyaert, J.; Efremov, R.; Ulens, C. (2016). Functional and biochemical characterization of Alvinella pompejana cys-loop receptor homologues. PLoS One 11(3): e0151183. https://dx.doi.org/10.1371/journal.pone.0151183
In: PLoS One. Public Library of Science: San Francisco. ISSN 1932-6203; e-ISSN 1932-6203, meer
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| Auteurs | | Top |
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- Pardon, E.
- Willegems, K.
- Bertrand, D.
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- Steyaert, J.
- Efremov, R.
- Ulens, C., meer
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| Abstract |
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 a 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. |
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