title: Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues.
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 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.
authors.affiliation: Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, University of Leuven, Leuven, Belgium.
authors.forename: Eveline
authors.initials: E
authors.lastname: Wijckmans
authors.affiliation: Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, University of Leuven, Leuven, Belgium.
authors.forename: Mieke
authors.initials: M
authors.lastname: Nys
authors.affiliation: Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, University of Leuven, Leuven, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: Debaveye
authors.affiliation: Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, University of Leuven, Leuven, Belgium.
authors.forename: Marijke
authors.initials: M
authors.lastname: Brams
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.|Structural Biology Research Center, VIB, Brussels, Belgium.
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.|Structural Biology Research Center, VIB, Brussels, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: Willegems
authors.affiliation: HiQscreen, Geneva, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Bertrand
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.|Structural Biology Research Center, VIB, Brussels, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.|Structural Biology Research Center, VIB, Brussels, Belgium.
authors.forename: Rouslan
authors.initials: R
authors.lastname: Efremov
authors.affiliation: Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, University of Leuven, Leuven, Belgium.
authors.forename: Chris
authors.initials: C
authors.lastname: Ulens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D058466:Cysteine Loop Ligand-Gated Ion Channel Receptors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D005998:Glycine / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007477:Ions; D008024:Ligands; D008969:Molecular Sequence Data; D011077:Polychaeta / Q000378:metabolism*; D055503:Protein Multimerization; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D061905:Single-Domain Antibodies / Q000378:metabolism; D013654:Taurine / Q000494:pharmacology; D013696:Temperature; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D058466:Cysteine Loop Ligand-Gated Ion Channel Receptors; D007477:Ions; D008024:Ligands; D021122:Protein Subunits; D020543:Proteome; D061905:Single-Domain Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013654:Taurine; D005680:gamma-Aminobutyric Acid; D005998:Glycine