An Internally Modulated, Thermostable, pH-sensitive Cys Loop Receptor from the Hydrothermal Vent Worm Alvinella pompejana

• Published in: The Journal of biological chemistry 2014-05, Vol.289 (21), p.15130-15140
• Main Authors: Juneja, Puneet, Horlacher, Reinhold, Bertrand, Daniel, Krause, Ryoko, Marger, Fabrice, Welte, Wolfram
• Format: Article
• Language: English
• Subjects: Alvinella pompejana; Amino Acid Sequence; Animals; Antiparasitic Agents - pharmacology; Base Sequence; Cys Loop Receptor; Cysteine Loop Ligand-Gated Ion Channel Receptors - classification; Cysteine Loop Ligand-Gated Ion Channel Receptors - genetics; Cysteine Loop Ligand-Gated Ion Channel Receptors - metabolism; Female; Hydrogen-Ion Concentration; Hydrothermal Vents; Ivermectin; Ivermectin - pharmacology; Membrane Biology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Membrane Proteins; Molecular Sequence Data; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation; Neurotransmitter Receptors; Nicotinic Acetylcholine Receptors; Oocytes - metabolism; Oocytes - physiology; Patch Clamp Electrophysiology; pH Sensitivity; Phylogeny; Picrotoxin - pharmacology; Polychaeta - genetics; Polychaeta - metabolism; Protein Conformation; Protein Stability; Recombinant Protein Expression; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Sf9 Cells; Temperature; Xenopus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.525576

Cys loop receptors (CLRs) are commonly known as ligand-gated channels that transiently open upon binding of neurotransmitters to modify the membrane potential. However, a class of cation-selective bacterial homologues of CLRs have been found to open upon a sudden pH drop, suggesting further ligands and more functions of the homologues in prokaryotes. Here we report an anion-selective CLR from the hydrothermal vent annelid worm Alvinella pompejana that opens at low pH. A. pompejana expressed sequence tag databases were explored by us, and two full-length CLR sequences were identified, synthesized, cloned, expressed in Xenopus oocytes, and studied by two-electrode voltage clamp. One channel, named Alv-a1-pHCl, yielded functional receptors and opened upon a sudden pH drop but not by other known agonists. Sequence comparison showed that both CLR proteins share conserved characteristics with eukaryotic CLRs, such as an N-terminal helix, a cysteine loop motif, and an intracellular loop intermediate in length between the long loops of other eukaryotic CLRs and those of prokaryotic CLRs. Both full-length Alv-a1-pHCl and a truncated form, termed tAlv-a1-pHCl, lacking 37 amino-terminal residues that precede the N-terminal helix, formed functional channels in oocytes. After pH activation, tAlv-a1-pHCl showed desensitization and was not modulated by ivermectin. In contrast, pH-activated, full-length Alv-a1-pHCl showed a marked rebound current and was modulated significantly by ivermectin. A thermostability assay indicated that purified tAlv-a1-pHCl expressed in Sf9 cells denatured at a higher temperature than the nicotinic acetylcholine receptor from Torpedo californica. Cys loop receptors from the hydrothermal vent worm Alvinella pompejana were studied. A Cys loop receptor opens at low pH, is chloride-specific, is modulated by its N-terminal extension, and is modestly thermostable. Cys loop receptors from worms living in extreme and secluded habitats are more closely related to eukaryotic than to prokaryotic family members. We studied the first known Cys loop receptor from a hydrothermal vent worm.