THE BINDING CHARACTERISTICS OF A HUMAN BLADDER RECOMBINANT P-2X PURINOCEPTOR, LABELED WITH [H-3] ALPHA-BETA-MEATP, [S-35] ATP-GAMMA-S OR [P-33] ATP

1 The binding of [H-3]-alpha beta meATP, [S-35]-ATP gamma S and [alpha (33)P]-ATP to a human bladder P-2X purinoceptor, transiently expressed in CHO-KI cells using the Semliki Forest Virus (SFV) expression syste m, was examined. The characteristics of the binding sites were compare d with results obtained in rat vas deferens, a tissue in which the rad ioligands are thought to label P-2X purinoceptors and in which the end ogenous P-2X purinoceptor displays high homology with the human bladde r P-2X purinoceptor. 2 In non-infected CHO-KI cells, 100 mu M ATP evok ed only small inward currents (40 pA) in approximately 30% of the cell s when studied by the whole-cell voltage clamp technique. In membranes prepared from either these non-infected cells or cells infected with SFV containing the LacZ gene (SFV-LacZ), [H-3]-alpha beta meATP bound with low affinity (pK(d)=7.04; B-max=8.88 pmol ml(-1) protein) and the re was only a low density of [S-35]-ATP gamma S binding sites (pK(d)=8 .74; B-max=358 fmol ml(-1) protein). These binding sites differed from those present in rat vas deferens. Thus: pIC(50) values for alpha bet a meATP (6.5) and L-beta gamma meATP (4.0) at the [H-3]-alpha beta meA TP binding sites in non-infected CHO-KI cells were much lower than the respective pIC(50) values of 8.3 and 7.7, determined in rat vas defer ens. Similarly, affinity estimates (PIC50 values) for A-TP (6.82), 2-m eS-ATP (5.43), ATP gamma S (7.06) and alpha beta meATP (4.84) at the [ S-35]-ATP gamma S binding sites in non-infected CHO-K1 cells were up t o 2291 fold lower than the respective values of 9.01, 8.79, 8.73 and 7 .57, determined in rat vas deferens. 3 In CHO-K1 cells infected using SFV containing the cDNA for the human bladder P-2X purinoceptor (SFV-h .P-2X), ATP, 2-meS-ATP and alpha beta meATP evoked large inward curren ts (2-7 nA) in whole cell voltage clamp studies. In membranes prepared from these SFV-h.P-2X infected cells, [H-3]-alpha beta meATP binding was increased, compared to that measured in the non infected or SFV-La cZ infected cells, with only high affinity [H-3]-alpha beta meATP bind ing sites being detected (pK(d)=9.21; B-max=3.54 pmol mg(-1) protein). The pIC(50) values for alpha beta meATP (8.2) and L-beta gamma meATP (7.2) in competing for these sites were the same or similar to the val ues determined in rat vas deferens. 4 A high density of [S-35]-ATP gam ma S binding sites (pK(d)=9.09; B-max=6.82 pmol mg(-1) protein) was al so present in the membranes from CHO-K1 cells infected with SFV-h.P-2X and affinity estimates (pIC(50) values) for ATP (8.93), 2-meS-ATP (8. 23), ATP gamma S (8.08), and alpha beta meATP (7.17) at competing for these sites were as much as 631 fold higher than the respective values determined in non-infected CHO-KI cells but were close to the values determined in rat vas deferens. Similar data were obtained with [alpha (33)P]ATP as radioligand. 5 These data suggest that [H-3]-alpha beta m eATP, [S-35]-ATP gamma S and [P-33]-ATP label the human bladder recomb inant P-2X purinoceptor expressed in CHO-KI cells following infection with SFV-h.P-2X and provide further corroborative evidence to support the contention that the high affinity binding sites for these radiolig ands in rat vas deferens are P-2X purinoceptors.