Effects of antagonists at the human recombinant P2X7 receptor

1 We have used whole-cell patch clamping methods to examine the properties of the recombinant human P2X(7) (P2Z) receptor stably expressed in HEK-293 cells. 2 In an extracellular solution with lowered concentrations of divalent cations (zero Mg2+ and 0.5 mM Ca2+), both ATP and the nucleotide analogue: 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (Bz-ATP) evoked concentration-dependent whole-cell inward currents with maxima of 4658+/-671 and 5385+/-990 pA, respectively, at a holding potential of -90 mV. Current-voltage relationships determined using 100 mu M Bz-ATP reversed at -2.7+/-3.1 mV, and did not display significant rectification. 3 Repeated applications of 300 mu M Bz-ATP produced inward currents with similar rise-times (approx. 450 ms, 5-95% current development) but with progressively slower 95-5% decay times, with the eighth application of this agonist yielding a decay time of 197% of the first application. 4 Concentration-effect curves to ATP and Bz-ATP produced estimated EC50 values of 780 and 52.4 mu M, respectively. Consecutive concentration-effect curves to Bz-ATP produced curves with similar maxima and EC50 values. 5 The non-selective P2 antagonists, pyridoxal-phosphate-6-azophenyl-, 2',4'-disulphonic acid (PPADS) and suramin, both produced concentration-dependent increases in maximal inward currents to Bz-ATP, with IC50 concentrations of approximately 1 mu M and 70 mu M, respectively. The profile of antagonism produced by PPADS was not that of a competitive antagonist. 6 The isoquinolene derivatives 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62) and calmidazolium both produced antagonism which was not competitive, with IC50 concentrations of approximately 15 and 100 nM, respectively. HMA (5-(N,N-hexamethylene)- amiloride) was also an effective antagonist at a concentration of 10 mu M. The group IIb metal, copper, also displayed antagonist properties at the human P2X(7) receptor, reducing the maximum response to Bz-ATP by about 50% at a concentration of 1 mu M. 7 These data demonstrate that the human recombinant P2X(7) receptor displays functional behaviour which is similar to the recombinant rat P2X(7) receptor, but has a distinct pharmacological profile with respect to agonist and antagonist sensitivity.