Cloning and pharmacological characterization of the dog P2X7 receptor

Abstract

Background and purpose: Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues. Experimental approach: A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1b release in dog and human whole blood. Key results: The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2′-&3′-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower. Conclusions and implications: Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic.

Mechanism of action of species‐selective P2X7 receptor antagonists

“…Studies on the human (Rassendren et al. , 1997), dog (Accession Number, EU334661.1; Roman et al. , 2007) and rat (Surprenant et al.…”

“…The nomenclature used for describing the P2X7 receptor conforms to the Journal's guidelines (Alexander et al, 2008). Studies on the human (Rassendren et al, 1997), dog (Acces-sion Number, EU334661.1; Roman et al, 2007) and rat (Surprenant et al, 1996) P2X 7 receptors were performed using HEK293 cells expressing the recombinant receptors (Fonfria et al, 2008) whereas studies on guinea-pig (Fonfria et al, 2008) and mouse (Young et al, 2006) P2X7 receptors were performed using the BacMam expression system previously described (Fonfria et al, 2008).…”

Insights into the Molecular Mechanisms Underlying Mammalian P2X7 Receptor Functions and Contributions in Diseases, Revealed by Structural Modeling and Single Nucleotide Polymorphisms

“…The P2X7Rs from six mammalian species (human, rhesus macaque monkey, dog, rat, mouse, and guinea pig) and two non-mammalian species ( Xenopus and zebrafish) have thus far been characterized in isolation to various extent (Surprenant et al, 1996; Chessell et al, 1997; Rassendren et al, 1997a; Paukert et al, 2002; Kucenas et al, 2003; Fonfria et al, 2008; Roman et al, 2009; Bradley et al, 2011b). As shown in Figure 1, all the mammalian P2X7R subunits comprise 595 amino acid residues except for the guinea pig P2X7R subunit, which is one residue shorter due to omission of Asp77 (in the human P2X7R numbering, used from this point onward).…”

The P2X7 Receptor Channel: Recent Developments and the Use of P2X7 Antagonists in Models of Disease

“…650 all cloned mammalian P2X7 receptors, except rat, when expressed in HEK-293 cells (Fonfria et al, 2008;Donnelly-Roberts et al, 2009;Roman et al, 2009;Bradley et al, 2011b). In comparison, the commonly used in vivo P2X7 antagonist BBG is at least a 3-fold more potent antagonist of rat, murine, canine, or guinea pig P2X7 than human P2X7 (Fonfria et al, 2008;Donnelly-Roberts et al, 2009;Roman et al, 2009).…”

“…The P2X7 receptor is encoded by the P2RX7 gene and belongs to the P2X family of trimeric ligand-gated cation channels, of which there are seven distinct members (P2X1-7) (Coddou et al, 2011). Of the P2X family, the P2X7 monomeric subunit is the largest, with a length of 595 amino acids for the human, rat, mouse, dog, and Rhesus macaque receptors (Surprenant et al, 1996;Rassendren et al, 1997;Chessell et al, 1998;Roman et al, 2009;Bradley et al, 2011b). Each subunit is characterized by relatively short and long intracellular amino and carboxyl (C) termini, respectively, as well as two hydrophobic membrane-spanning segments (transmembrane domains) separated by a long glycosylated extracellular ATP-binding domain.…”