Effects of antagonists at the human recombinant P2X 7 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 Mg 2+ and 0.5 m m Ca 2+ ), 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 μ m Bz‐ATP reversed at −2.7±3.1 mV, and did not display significant rectification. 3 Repeated applications of 300 μ 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 EC 50 values of 780 and 52.4 μ m , respectively. Consecutive concentration‐effect curves to Bz‐ATP produced curves with similar maxima and EC 50 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 IC 50 concentrations of approximately 1 μ m and 70 μ 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 IC 50 concentrations of approximately 15 and 100 n m , respectively. HMA (5‐(N,N‐hexamethylene)‐ amiloride) was also an effective antagonist at a concentration of 10 μ 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 μ 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.British Journal of Pharmacology (1998) 124 , 1314–1320; doi: 10.1038/sj.bjp.0701958