Pharmacological and genetic characterisation of the canine P2X4 receptor

Authors

Reece Sophocleous, University of WollongongFollow
Tracey Berg, University of WollongongFollow
Rocio K. Finol Urdaneta, University of WollongongFollow
Vanessa Sluyter, University Of Wollongong
Shikara Keshiya, University of Wollongong
Lachlan Bell, University of Wollongong
Stephen Curtis
Belinda Curtis
Aine Seavers
Rachael Bartlett, University of WollongongFollow
Mark P. Dowton, University of WollongongFollow
Leanne Stokes
Lezanne Ooi, University of WollongongFollow
Ronald Sluyter, University of WollongongFollow

RIS ID

142087

Publication Details

Sophocleous, R., Berg, T., Finol Urdaneta, R., Sluyter, V., Keshiya, S., Bell, L., Curtis, S., Curtis, B., Seavers, A., Bartlett, R., Dowton, M., Stokes, L., Ooi, L. & Sluyter, R. (2020). Pharmacological and genetic characterisation of the canine P2X4 receptor. British Journal of Pharmacology,

Abstract

© 2020 The British Pharmacological Society Background and Purpose: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. Experimental Approach: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. Key Results: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2′(3′)-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. Conclusion and Implications: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans.