Background and purpose: Several P2X 7 receptor antagonists are allosteric inhibitors and exhibit species difference in potency. Furthermore, N 2 -(3,4-difluorophenyl)-N 1 -(2-methyl-5-(1-piperazinylmethyl)phenyl)glycinamide dihydrochloride (GW791343) exhibits negative allosteric effects at the human P2X 7 receptor but is a positive allosteric modulator of the rat P2X 7 receptor. In this study we have identified several regions of the P2X 7 receptor that contribute to the species differences in antagonist effects. Experimental approach: Chimeric human-rat P2X 7 receptors were constructed with regions of the rat receptor being inserted into the human receptor. Antagonist effects at these receptors were measured in ethidium accumulation and radioligand binding studies. Key results: Exchanging regions of the P2X 7 receptor close to transmembrane domain 1 modified the effects of KN62, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and GW791343. Further studies, in which single amino acids were exchanged, identified amino acid 95 as being primarily responsible for the differential allosteric effects of GW791343 and, to varying degrees, the species differences in potency of SB203580 and KN62. The species selectivity of pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulphonic acid was affected by multiple regions of the receptor, with potency being particularly affected by the amino acid 126 but not by amino acid 95. A further region of the rat receptor (amino acids 154-183) was identified that, when inserted into the corresponding position in the human receptor, increased ATP potency 10-fold. Conclusions: This study has identified several key residues responsible for the species differences in antagonist effects at the P2X 7 receptor and also identified a further region of the P2X 7 receptor that can significantly affect agonist potency at the P2X 7 receptor.
Background: Recent studies have implicated the mitogen activated protein kinase (MAPK) in cellular permeability changes following P2X 7 receptor activation in native tissues. In this study we have further studied the effect of MAPK inhibitors on recombinant and native P2X 7 receptors. Experimental Approach: The MAPK inhibitors SB-203580, SB-202190 and SB-242235 were examined in HEK293 cells expressing recombinant P2X 7 receptors and in THP-1 cells expressing native human P2X 7 receptors using a range of experimental approaches. Key results: At human recombinant P2X 7 receptors, SB-203580 and SB-202190 were weak, non-competitive inhibitors (pIC 50 录 4.8 -6.4) of ethidium accumulation stimulated by 2'-& 3'-O-(4benzoylbenzoyl)-ATP (BzATP) but SB-242235 (0.1-10mM) had no effect. SB-203580 and SB-202190 had no effect on rat or mouse recombinant P2X 7 receptors and studies with chimeric P2X 7 receptors suggested that SB-203580 was only effective in chimeras containing the N-terminal 255aa of the human P2X 7 receptor. SB-203580 did not consistently affect BzATP-mediated increases in cell calcium levels and, in electrophysiological studies, it slightly decreased responses to 30mM BzATP but potentiated responses to 100mM BzATP. In THP1 cells, SB-203580 modestly inhibited BzATP-stimulated ethidium accumulation (pIC 50 5.7 -o5) but SB-202190 had no effect. Finally, SB-203580 did not block BzATP-stimulated interleukin-1b release in THP-1 cells. Conclusions: This study confirms that high concentrations of SB-203580 and SB-202190 can block human P2X 7 receptormediated increases in cellular ethidium accumulation but suggest this is not related to MAPK inhibition. Overall, the data cast doubt on a general role of MAPK in mediating P2X 7 receptor mediated changes in cellular permeability.
In this study we have expressed recombinant P2X7 receptors in HEK293 cells and examined the reasons for the species- and agonist-dependent differences in the time taken for the closure of the P2X7 receptor ion-channels after agonist removal. Channel closure times, measured in electrophysiological studies or by measuring cellular permeability to ethidium cations, were slower at rat than at human or mouse P2X7 channels following washout of the P2X7 agonist 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP). In contrast, there were no species differences in channel closure times when ATP was the agonist. BzATP was more potent than ATP at the three species homologues and exhibited highest potency for rat P2X7 receptors suggesting that channel closure time was related to agonist potency. Furthermore, BzATP potency for the P2X7 receptor could be modified by changing extracellular ionic concentrations or by mutating the receptor and modifications which increased agonist potency also increased the time taken for channel closure. The dependence of channel closure time on agonist potency suggests it reflects agonist dissociation from the P2X7 receptor rather being an intrinsic property of the ion-channel. Consistent with this, our previous studies have shown that agonist potency increases after repeated agonist applications and in this study channel closure time at rat P2X7 receptors increased after repeated agonist applications. Overall these results suggest that the species differences in channel closure times reflect differences in agonist dissociation rates which arise as a consequence of the marked species differences in agonist potency.
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