Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.
OBJECTIVE
The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor.


METHODS
The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]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 P2X(7) receptors.


RESULTS
Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) 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 P2X(7) 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 P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.
Michel
A D
AD
Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research and Development Limited, Harlow, Essex, UK. adm7393@gsk.com
Chambers
L J
LJ
Clay
W C
WC
Condreay
J P
JP
Walter
D S
DS
Chessell
I P
IP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D002478:Cells, Cultured; D006801:Humans; D007472:Iohexol / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D011869:Radioligand Assay / Q000379:methods*; D018048:Receptors, Purinergic P2 / Q000032:analysis* / Q000378:metabolism; D058486:Receptors, Purinergic P2X7; D014316:Tritium; D014638:Vanadates / Q000494:pharmacology
C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D014316:Tritium; C012622:compound 17; D014638:Vanadates; D007472:Iohexol; D000255:Adenosine Triphosphate