Abstract
The P2X4 receptor is an ATP-gated ion channel that was proposed as a novel drug target for neuropathic pain, neurodegenerative diseases, epilepsy, and cancer. Animal models are indispensable for elucidating the role of receptors in health and disease and for drug development. Here, we present a systematic analysis and comparison of P2X4 receptors of nine different species that are relevant for basic and applied research, human, monkey, dog, guinea pig, pig, rabbit, rat, mouse, and zebrafish. 1321N1 astrocytoma cell lines stably expressing the respective receptor were generated by retroviral transfection. Calcium influx assays were performed to assess the effects of structurally diverse P2X4 receptor agonists and antagonists with the aim of exploring species differences. Most agonists showed significantly reduced potency in mice, rats, and zebrafish in comparison to the human P2X4 receptor. The physiological agonist ATP was more potent at monkey, rabbit, guinea pig, and pig P2X4 receptors (0.0285-0.0594 μM) than at the human ortholog (0.269 μM) and least potent at the zebrafish receptor (3.27 mM). 2-MeS-ATP exhibited similar potency (0.290-4.50 μM) in all investigated P2X4 receptor species except zebrafish and is therefore a suitable tool compound for studying P2X4 receptors across species. The allosteric P2X4 receptor antagonists BX430 and 5-BDBD displayed notable species differences, while the indole derivative PSB-OR-2020 blocked all investigated mammalian species with high potency (0.696-6.32 nM), exhibiting only minor species differences. These data will be valuable to support future in vitro and, in particular, in vivo studies.