Abstract
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.
Highlights
Cys loop receptors from the hydrothermal vent worm Alvinella pompejana were studied
A multiple alignment of the Alv-a1-pHCl constructs and Alv-a9 with members of the Cys loop receptors (CLRs) family is given in supplemental Fig. 1A
Alv-a9 shows 27.0% identity with the human nicotinic acetylcholine receptor (nAChR) ␣9 subunit (Fig. 2, A and B). Both were predicted with individual signal sequences of 25 and 18 residues and an N-terminal helix followed by LBDs (Fig. 2), as is distinctive for eukaryotic CLRs
Summary
Cys loop receptors from the hydrothermal vent worm Alvinella pompejana were studied. We report an anion-selective CLR from the hydrothermal vent annelid worm Alvinella pompejana that opens at low pH. One channel, named Alv-a1-pHCl, yielded functional receptors and opened upon a sudden pH drop but not by other known agonists. 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 GLIC for instance shares 20% sequence identity with the human ␣7 nicotinic acetylcholine receptor (nAChR)) and is made up of a smaller number of residues [7] This is mainly due to the absence of the N-terminal helix and a much shorter M3-M4 linker [8].
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