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BMC Evol Biol
2009 Jan 20;9:17. doi: 10.1186/1471-2148-9-17.
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A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper.
Bavan S
,
Straub VA
,
Blaxter ML
,
Ennion SJ
.
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BACKGROUND: Orthologs of the vertebrate ATP gated P2X channels have been identified in Dictyostelium and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including Drosophila melanogaster and Caenorhabditis elegans, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm Schistosoma mansoni have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (HdP2X) from the tardigrade Hypsibius dujardini demonstrating that purinergic signalling is preserved in some ecdysozoa.
RESULTS: ATP (EC50 approximately 44.5 microM) evoked transient inward currents in HdP2X with millisecond rates of activation and desensitisation. HdP2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC50 15.0 microM) and suramin (IC50 22.6 microM) and zinc and copper inhibit ATP-evoked currents with IC50 values of 62.8 microM and 19.9 microM respectively. Site-directed mutagenesis showed that unlike vertebrate P2X receptors, extracellular histidines do not play a major role in coordinating metal binding in HdP2X. However, H306 was identified as playing a minor role in the actions of copper but not zinc. Ivermectin potentiated responses to ATP with no effect on the rates of current activation or decay.
CONCLUSION: The presence of a P2X receptor in a tardigrade species suggests that both nematodes and arthropods lost their P2X genes independently, as both traditional and molecular phylogenies place the divergence between Nematoda and Arthropoda before their divergence from Tardigrada. The phylogenetic analysis performed in our study also clearly demonstrates that the emergence of the family of seven P2X channels in human and other mammalian species was a relatively recent evolutionary event that occurred subsequent to the split between vertebrates and invertebrates. Furthermore, several characteristics of HdP2X including fast kinetics with low ATP sensitivity, potentiation by ivermectin in a channel with fast kinetics and distinct copper and zinc binding sites not dependent on histidines make HdP2X a useful model for comparative structure-function studies allowing a better understanding of P2X receptors in higher organisms.
Figure 1. Amino acid alignment of HdP2X. The predicted amino acid sequence of HdP2X was aligned with human P2X1, and 4, rat P2X2 and non vertebrate P2X channels from S. mansoni (smP2X) [12]O. tauri (OtP2X) [11], M. brevicollis (MbP2X) [11] and D. discoideum (dP2XA) [10]. Predicted transmembrane regions in HdP2X (TopPred algorithm [19]) are depicted by black horizontal lines. Functionally important amino acid residues using human P2X1 residue numbering (indicated by *) are highlighted in bold above the sequence including positively charged lysine residues, FT and NFR motifs, conserved cysteines and a conserved consensus protein kinase C phosphorylation site. Point mutations made in HdP2X to investigate zinc and copper binding are indicated in bold below the sequence.
Figure 2. Phylogenetic relationships of Hypsibius dujardini P2X. Aligned protein sequences from P2X homologues from H. dujardini (Hd) (Tardigrada), vertebrates (represented by human (h), mouse (m) and rat (r); these taxa have seven paralogous P2X genes (P2X1–7)), S. mansoni (Sm) (Platyhelminthes), L. stagnalis (Lym) (Mollusca), M. brevicollis (Mb) (Choanoflagellida), D. discoideum (d) (Mycetozoa; Dictyosteliida; five paralogues) and two Ostreococcus species, O. tauri (Ot) and O. lucimarinus (Ol) (Chlorophyta; Prasinophyceae) were analysed using Neighbour Joining and maximum parsimony methods. Both methods agreed on the well supported nodes figured in this phylogram. Nodes were supported with bootstraps of 100% unless otherwise indicated; all nodes with less than 75% bootstrap support were collapsed.
Figure 3. Properties of ATP evoked currents. Two-electrode voltage clamp recordings at a holding membrane potential of -60 mV were made from oocytes expressing HdP2X. A. Comparison of HdP2X and human P2X1 receptors. Currents were recorded in response to 100 μM ATP, indicated by bar. HdP2X displays faster current rise and decay times than the human P2X1 and displays faster recovery from desensitisation between the first and second applications of agonist (applications 5 minutes apart). B. Current voltage relationship of HdP2X. The reversal potential of ATP mediated currents was determined by recording ATP (100 μM, indicated by bar) induced currents at holding potentials ranging from -100 mV to +40 mV with a 5 minute interval between applications. Currents obtained in different oocytes were normalised to the current obtained at -60 mV for each individual cell (n = 6). C. Example currents for the plot depicted in B. D. Concentration response curves for ATP, Bz-ATP and αβ-me-ATP. Mean currents were normalised to the response given by 100 μM ATP (n = 5–7). E and F. Example currents recorded in response to ATP (E) and Bz-ATP (F) (concentrations in μM, agonist application indicated by bar).
Figure 4. HdP2X is antagonised by suramin and PPADS. The effects of the P2 receptor antagonists suramin and PPADS were determined in Xenopus oocytes expressing HdP2X at a holding membrane potential of -60 mV. A. Example currents in response to 100 μM ATP in the presence and absence of 100 μM suramin (five minutes between sequential applications). B. Example currents in response to 100 μM ATP in the presence and absence of 100 μM PPADS (five minutes between sequential applications). C. Inhibition curves for mean responses to 100 μM ATP in the presence of suramin (closed squares) (n = 5) and PPADS (open circles) (n = 5).
Figure 5. HdP2X receptor currents are inhibited by zinc and copper. Two-electrode voltage clamp recordings were made from Xenopus oocytes expressing HdP2X at a holding potential of -60 mV. A. Concentration response curves for 100 μM ATP (~EC80) in the presence of varying concentrations of zinc (open circles) (n = 6) and copper (closed squares) (n = 6). B. Effects of single and double point mutations on inhibition by 100 μM zinc and copper. Mean currents are normalised to the 100 μM ATP response in the absence of zinc or copper for wild type and mutant receptors (n = 7). C. ATP concentration response curves for histidine point mutations (n = 7). For each single and double histidine mutation no significant change in ATP sensitivity was observed suggesting that mutation had not resulted in gross changes in receptor conformation. The triple histidine mutation H232A/H252A/H306A produced a non functional channel and so could not be studied.
Figure 6. Ivermectin potentiates ATP evoked currents at HdP2X. Two-electrode voltage clamp recordings were made from Xenopus oocytes expressing HdP2X. A. Representative ATP (100 μM) evoked currents recorded from the same cell in the presence and absence of 3 μM ivermectin (ATP/ivermectin application indicated by bars). Five minutes recovery was allowed between applications and ivermectin was bath perfused at 3 μM in the five minutes preceding the second recording as well as being present in the ATP application. B. Concentration response curves for ATP in the presence (open circles) and absence (closed squares) of 3 μM ivermectin (n = 7). Mean currents were normalised to the response produced by 100 μM ATP in the absence of ivermectin.
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