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 and purpose: The human, rat, and mouse P2X 7 receptors have been previously characterized, and in this study we report the cloning and pharmacological properties of the guinea pig orthologue. Experimental approach: A cDNA encoding for the guinea pig P2X 7 receptor was isolated from a guinea pig brain library. The receptor was expressed in U-2 OS cells using the BacMam viral expression system. A monoclonal antibody was used to confirm high levels of cell surface expression and the functional properties were determined in ethidium bromide accumulation studies. Key results: The predicted guinea pig protein is one amino acid shorter than the human and rat orthologues and over 70% identical to the rat and human receptors. In contrast to human and rat P2X 7 receptors, 2 0 -&3 0 -O-(4benzoylbenzoyl)ATP (BzATP) was a partial agonist of the guinea pig P2X 7 receptor when compared to ATP and acted as an antagonist in some assays. However, as at other species orthologues, BzATP was more potent than ATP. The guinea pig P2X 7 receptor possessed higher affinity for 1-[N,O-bis(5-isoquinoline sulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62), suramin and Coomassie Brilliant Blue than human or rat P2X 7 receptors suggesting that it is pharmacologically different to other rodent or human P2X 7 receptors. Conclusions and implications: The guinea pig recombinant P2X 7 receptor displays a number of unique properties that differentiate it from the human, rat and mouse orthologues and this structural and functional information should aid in our understanding of the interaction of agonists and antagonist with the P2X 7 receptor.
Background and purpose: AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl] phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X7 receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues. Experimental approach: Antagonist effects at recombinant and chimeric P2X7 receptors were assessed by ethidium accumulation and radioligand-binding studies. Key results: AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X7 receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X7 receptors and, for each compound, their potency estimates at human and dog P2X7 receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X7 receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X7 receptor. Conclusions: AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X7 receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X7 receptor at which antagonists can bind and affect receptor function.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.