A P2X receptor from the tardigrade species Hypsibius dujardiniwith fast kinetics and sensitivity to zinc and copper

AbstractBackgroundOrthologs of the vertebrate ATP gated P2X channels have been identified inDictyosteliumand 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 includingDrosophila melanogasterandCaenorhabditis elegans, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatwormSchistosoma mansonihave 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 tardigradeHypsibius dujardinidemonstrating that purinergic signalling is preserved in some ecdysozoa.ResultsATP (EC50~44.5 μM) evoked transient inward currents inHdP2X with millisecond rates of activation and desensitisation.HdP2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC5015.0 μM) and suramin (IC5022.6 μM) and zinc and copper inhibit ATP-evoked currents with IC50values 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 inHdP2X. 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 ofHdP2X 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 makeHdP2X a useful model for comparative structure-function studies allowing a better understanding of P2X receptors in higher organisms.