Functional characterization of the P2X(4) receptor orthologues

1 The aim of this study was to functionally characterize the recombinant mo use P2X(4) receptor and to compare its pharmacological properties with thos e of the human and rat orthologues. 2 Whole cell recordings were made from rafts of HEK-293 cells stably expres sing recombinant mouse, rat or human P2X(4) receptors. using Cs-aspartate c ontaining electrodes (3-8 M Omega) in a HEPES-buffered extracellular medium . 3 The agonist potency of ATP at the three species orthologues was similar, with mean ECS, values of 2.3 mu M 1.4 mu M and 5.5 mu M, respectively. 4 Adenosine-5'-tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X(4) receptors (EC50 = 2.6 and 3.0 mu M), but was significantly less potent at the rat orthologue (EC50 = 20.0 mu M). alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) also act ed as a partial agonist, producing 29% of the maximum response at the mouse P2X(4) and 24% at the human P2X(4) receptor. 5 In contrast to the other species orthologues. alpha,beta-meATP failed to elicit a significant agonist response at rat P2X(4) receptors, and was foun d to act as an antagonist, with an IC50, of 9.6 mu M, against 10 mu M ATP. 6 Mouse P2X(4) receptors were found to be sensitive to the antagonist, pyri doxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (IC50 = 10.5 mu M ), as were human P2X(4) receptors (IC50 = 9.6 mu M). The rat receptor howe ver, showed a low sensitivity to PPADS (IC50 > 100 mu M). 7 All three orthologues were relatively suramin-insensitive (IC50 > 100 mu M) and insensitive to 1-[N,O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phen ylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62; IC50 > 3 mu M). 8 Our results suggest that the pharmacological properties of the mouse rece ptor ale most similar to the human P2X(4) receptor, and differ markedly fro m the rat receptor.