An internally modulated, thermostable, pH-sensitive Cys loop receptor from the hydrothermal vent worm Alvinella pompejana.
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.
From the Fachbereich Biologie, Universität Konstanz, Universitätsstraβe 10, 78457 Konstanz, Germany.
Puneet
P
Juneja
Trenzyme, Byk-Gulden-Straβe 2, 78467 Konstanz, Germany, and.
Reinhold
R
Horlacher
HiQScreen Sàrl, 6, rte. de Compois, 1222 Vésenaz, Geneva, Switzerland.
Daniel
D
Bertrand
HiQScreen Sàrl, 6, rte. de Compois, 1222 Vésenaz, Geneva, Switzerland.
Ryoko
R
Krause
HiQScreen Sàrl, 6, rte. de Compois, 1222 Vésenaz, Geneva, Switzerland.
Fabrice
F
Marger
From the Fachbereich Biologie, Universität Konstanz, Universitätsstraβe 10, 78457 Konstanz, Germany, wolfram.welte@uni-konstanz.de.
Wolfram
W
Welte
D000595:Amino Acid Sequence; D000818:Animals; D000977:Antiparasitic Agents / Q000494:pharmacology; D001483:Base Sequence; D058466:Cysteine Loop Ligand-Gated Ion Channel Receptors / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D060086:Hydrothermal Vents; D007559:Ivermectin / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D010802:Phylogeny; D010852:Picrotoxin / Q000494:pharmacology; D011077:Polychaeta / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D061987:Sf9 Cells; D013696:Temperature; D014981:Xenopus
D000977:Antiparasitic Agents; D058466:Cysteine Loop Ligand-Gated Ion Channel Receptors; D007559:Ivermectin; D050505:Mutant Proteins; D010852:Picrotoxin
Alvinella pompejana; Cys Loop Receptor; Ivermectin; Membrane Proteins; Neurotransmitter Receptors; Nicotinic Acetylcholine Receptors; Patch Clamp Electrophysiology; Protein Conformation; Recombinant Protein Expression; pH Sensitivity