An internally modulated, thermostable, pH-sensitive Cys loop receptor from the hydrothermal vent worm Alvinella pompejana

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.

Keywords: Alvinella pompejana; Cys Loop Receptor; Ivermectin; Membrane Proteins; Neurotransmitter Receptors; Nicotinic Acetylcholine Receptors; Patch Clamp Electrophysiology; Protein Conformation; Recombinant Protein Expression; pH Sensitivity.

FIGURE 1.

Different expression constructs of Alv-a1-phCl. Alv-a1-pHCl has an N-terminal extension of 37 residues (supplemental Fig. 1A) followed by the N-terminal helix and the LBD. The transmembrane part is formed by the transmembrane helices M1-M4. A, Alv-a1-pHCl full-length protein. B, tAlv-a1-phCl lacks 37 N-terminal residues that likely are disordered. C, tAlv-a1-phCl-AGT. The M3-M4 loop is replaced by the tripeptide AGT. D, thAlv-a1-pHCl, N-terminally truncated by the 37 N-terminal residues and the N-terminal helix. E, thAlv-a1-pHCl-AGT, as the latter, but with the M3-M4 helix replaced by the tripeptide AGT. The constructs in A–C were used for oocyte experiments, and the constructs in B–E were used for Sf9 expression.

FIGURE 2.

Sequence alignments of Alv-a1-pHCl and Alv-a9 with their closest homologues. Alignment of Alv-a1-pHCl with the glutamate-gated ion channel of C. elegans and human glycine α1 (A) and Alv-a9 with the human α9 sequence (B). The construct tAlv-a1-pHCl is N-terminally truncated before Ser-65 (green, boxed). The M3-M4 loop is marked in pink. Secondary structure elements are indicated by bars. An alignment with all members of the CLR family is given in supplemental Fig. 1A.

FIGURE 3.

Relationship with other members of the CLR family. The dendrogram was constructed using a phylogenetic analysis platform. Alignment was done with MUSCLE (47) and phylogenetic analysis by PHYML (13, 45), with a statistical set of bootstrap values of 100 (46).

FIGURE 4.

Thermostability assay for tAlv-a1-pHCl and nAChR from T. californica. The Torpedo receptor was affinity-purified in complex with bungarotoxin in the detergent Cymal6. The tAlv-a1-pHCl receptor was solubilized with DDM and purified as described above. Torpedo nAChR was incubated for 10 min at 50 °C (lane 1) and at room temperature (lane 2). Alv-a1-pHCl was incubated for 10 min at room temperature and at 40, 50, 55, 65, and 70 °C (lanes 4–9) and for 20 min at 50 °C (lane 10). The samples were applied to a blue native PAGE with a 18–4% acrylamide gradient. The marker proteins (lane 3) were at 670, 440, 230, and 140 kDa.

FIGURE 5.

Channel activity of Alv-a1-pHCl and tAlv-a1-pHCl observed in Xenopus oocytes. A, tAlv-a1-pHCl was expressed in Xenopus oocytes, and activation was tested with different neurotransmitters (the concentrations tested are indicated). Robust inward currents were only evoked by lowering the pH. B, the same for Alv-a1-pHCl. Control experiments with non-expressing oocytes were conducted at pH 4.7 (47 oocytes) and pH 3.0 (66 oocytes) and yielded average currents of −0.12 and −0.40 μA with a standard error of 0.09 and 0.20, respectively.

FIGURE 6.

Activation of tAlv-a1-pHCl by pH. A, tAlv-a1-pHCl course pH screen. B, tAlv-a1-pHCl fine pH screen. C, pH induced channel activation was fitted with a Hill equation and yielded an EC₅₀ of 3.24 and a Hill coefficient of 2.5 for tAlv-a1-pHCl. Error bars indicate mean ± S.E. The currents measured at the indicated pH values are from between 4 and 14 oocytes.

FIGURE 7.

tAlv-a1-pHCl is permeable to chloride ions. The reversal potential for the pH-evoked current was determined by applying a voltage ramp protocol, illustrated by the blue traces in the top panel, first under control conditions (87.6 mm) and then in a series of decreasing extracellular concentrations of chloride. In these experiments, sodium chloride was replaced by mannitol. Reducing the extracellular chloride concentration caused both a reduction in the amplitude of the response, as seen on the current traces, and a shift of the reversal potential observed in the center panel. The plot of the shift of the reversal potential as a function of the logarithm of the extracellular chloride concentration reveals that the Alv-a1-pHCl receptors are permeable to chloride ions and display an ionic selectivity close to the 58 mV/decade predicted for a channel exclusively permeable to chloride ions. Error bars indicate mean ± S.E.

FIGURE 8.

tAlv-a1-pHCl is partially inhibited in the presence of picrotoxin. A, data obtained from a single cell with exposure to different concentrations of picrotoxin (PTX) illustrates that these molecules partially inhibit the pH-evoked response. Cells were exposed for 10 s in the presence of picrotoxin before the pH jump (pH 3). B, plot of the current, normalized to unity, versus the response for picrotoxin recorded under control conditions yielded a dose-response curve that is fitted with a Hill equation with an IC₅₀ of 123 μm and a coefficient of 0.8. The data are from three oocytes. Error bars indicate mean ± S.E. Note that even at the highest concentration tested (3200 μm), picrotoxin only partially inhibits the pH-evoked current, and a constant of 0.22 was added.

FIGURE 9.

tAlv-a1-pHCl is insensitive to the modulators ivermectin and PNU-120596. Shown is the current after a pH drop from 7.5 to 3.5 (black bar). Green, without exposure; red, pre-exposure with 10 μm ivermectin; blue, pre-exposure with 10 μm PNU-120596.

FIGURE 10.

Alv-a1-pHCl shows a rebound current upon activation and is coactivated by ivermectin. A, green, current after a pH drop 7.5 to 3.5 (black bar). B, red, current from the same oocyte after ivermectin exposure (10 μm) for 60 s (red bar), washing, and a pH drop as above. This experiment was repeated with four different oocytes, yielding very similar currents.