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2020 - Pharmacological and genetic characterisation of the canine P2X4 receptor

icon pl   Patchliner publication in British Journal of Pharmacology (2020)

Authors:
Sophocleous R.A., Berg, T., Finol‐Urdaneta R.K., Sluyter V., Keshiya S., Bell L., Curtis S.J., Curtis B.L., Seavers A., Bartlett R., Dowton M., Stokes L., Ooi L., Sluyter R.

Journal: 
British Journal of Pharmacology (2020) doi: 10.1111/bph.15009


Abstract: 

Background and Purpose
The P2X4 receptor is an emerging therapeutic target for the treatment of chronic pain and cardiovascular disease. Dogs are well‐recognised natural models of human disease but information regarding P2X4 in dogs is absent. To aid the development and validation of P2X4‐targeting therapeutics, this study aimed to characterise and compare canine and human P2X4.

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 tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. P2X4 pharmacology was characterised using nucleotide‐induced Fura‐2 AM measurements of intracellular Ca2+ responses and known P2X4 antagonists in 1321N1 and HEK293 cells. P2X4‐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 were localised primarily to lysosomal compartments. ATP was identified as the primary agonist of canine P2X4 with near identical efficacy and potency at human P2X4. 2’(3’)‐O‐(4‐benzoyl)benzoyl ATP (BzATP), but not ADP, was identified as a partial agonist with reduced potency for dog P2X4 compared to the human orthologue. Five antagonists inhibited canine P2X4, with BX430 displaying reduced sensitivity and potency against canine P2X4.

Conclusion and Implications
P2X4 is highly conserved across dog pedigrees and displays a similar expression pattern and pharmacological profile to human P2X4, providing support for validation and use of therapeutics designed for P2X4‐related disease onset and management in dogs and humans.


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