Abstract
BackgroundOrthologs 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.ResultsATP (EC50 ~44.5 μM) 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 μM) and suramin (IC50 22.6 μM) and zinc and copper inhibit ATP-evoked currents with IC50 values of 62.8 μM and 19.9 μM 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.ConclusionThe 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.
Highlights
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
The aim of this present work was to determine whether the Hypsibius dujardini expressed sequence tag (EST) sequence corresponds to a gene encoding a functional P2X receptor and to determine its pharmacological properties. This was achieved by expression of cRNA encoding the Hypsibius dujardini P2X gene in Xenopus oocytes to enable two electrode voltage clamp recordings of ATP evoked membrane currents. Using this approach we show that the Hypsibius dujardini EST sequence does code a functional P2X receptor with fast activation and desensitisation kinetics and similar to some vertebrate P2X channels, is potentiated by ivermectin and inhibited by zinc and copper
HdP2X contains a number of conserved features typical to P2X channels including ten cysteine residues in the extracellular domain [2], a consensus protein kinase C phosphorylation site in the amino terminal [20], and the lysine residues and NFR/FT motifs shown to be involved in agonist binding (Fig. 1) [21]
Summary
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. Many studies have described potential roles for ATP as an extracellular signalling molecule in a range of invertebrate phyla [6] and plants [7,8] leading to the assumption that ATP is a primitive signalling molecule and that the emergence of purinergic receptors occurred relatively early in evolution [9]. This assumption is supported by the definitive molecular and functional identification of P2X receptors in the slime mould Dictyostelium discoideum [10], the green alga Ostreococcus tauri [11] and the choanoflagellate Monosiga brevicollis [11]. Given that P2X receptors are present in choanoflagellates, believed to be the sister group to the Metazoa, and in some representatives of the protostome superphylum Lophotrochozoa including Schistosoma mansoni [12] and Lymnaea stagnalis [13], the absence of P2X receptors from the above fully sequenced nematode and arthropod genomes suggests a loss of this class of gene in an ancestor of the Ecdysozoa
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