Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]‐labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N‐[2‐({2‐[(2‐hydroxyethyl)amino]ethyl}amino)‐5‐quinolinyl]‐2‐tricyclo[3.3.1.13,7]dec‐1‐ylacetamide (compound‐17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U‐2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]‐compound‐17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound‐17 and enhancement of radioligand binding by KN62 and unlabelled compound‐17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound‐17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound‐17 bind at separate, interacting, sites on the P2X7 receptor. British Journal of Pharmacology (2007) 151, 84–95. doi:10.1038/sj.bjp.0707196