Functional characterization of the P2X4 receptor orthologues

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Abstract
The aim of this study was to functionally characterize the recombinant mouse P2X4 receptor and to compare its pharmacological properties with those of the human and rat orthologues. Whole cell recordings were made from rafts of HEK‐293 cells stably expressing recombinant mouse, rat or human P2X4 receptors, using Cs‐aspartate containing electrodes (3–8 MΩ) in a HEPES‐buffered extracellular medium. The agonist potency of ATP at the three species orthologues was similar, with mean EC50 values of 2.3 μM, 1.4 μM and 5.5 μM, respectively. Adenosine‐5′‐tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X4 receptors (EC50=2.6 and 3.0 μM), but was significantly less potent at the rat orthologue (EC50=20.0 μM). α,β‐methylene adenosine‐5′‐triphosphate (α,β‐meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X4 and 24% at the human P2X4 receptor. In contrast to the other species orthologues, α,β‐meATP failed to elicit a significant agonist response at rat P2X4 receptors, and was found to act as an antagonist, with an IC50 of 4.6 μM, against 10 μM ATP. Mouse P2X4 receptors were found to be sensitive to the antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (IC50=10.5 μM), as were human P2X4 receptors (IC50=9.6 μM). The rat receptor however, showed a low sensitivity to PPADS (IC50>100 μM). All three orthologues were relatively suramin‐insensitive (IC50>100 μM) and insensitive to 1‐[N,O‐Bis(5‐isoquinoline sulphonyl)benzyl]‐2‐(4‐phenylpiperazine)ethyl]‐5‐isoquinoline sulphonamide (KN‐62; IC50>3 μM). Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X4 receptor, and differ markedly from the rat receptor. British Journal of Pharmacology (2000) 129, 388–394; doi:10.1038/sj.bjp.0703059