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 P2X7 (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 microM Bz-ATP reversed at -2.7+/-3.1 mV, and did not display significant rectification. 3. Repeated applications of 300 microM 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 microM, 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 microM and 70 microM, 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-phenylpi perazine (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 microM. The group IIb metal, copper, also displayed antagonist properties at the human P2X7 receptor, reducing the maximum response to Bz-ATP by about 50% at a concentration of 1 microM. 7. These data demonstrate that the human recombinant P2X7 receptor displays functional behaviour which is similar to the recombinant rat P2X7 receptor, but has a distinct pharmacological profile with respect to agonist and antagonist sensitivity.
Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge.
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Chessell
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Michel
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Humphrey
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D058919:Purinergic P2 Receptor Antagonists; D058486:Receptors, Purinergic P2X7; D011994:Recombinant Proteins / Q000819:agonists
C546468:P2RX7 protein, human; C546470:P2rx7 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D058486:Receptors, Purinergic P2X7; D011994:Recombinant Proteins; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate