title: Inhibition mechanism of the recombinant rat P2X(2) receptor in glial cells by suramin and TNP-ATP.
abstract: P2X receptors play an important role in communication between cells in the nervous system. Therefore, understanding the mechanisms of inhibition of these receptors is important for the development of new tools for drug discovery. Our objective has been to determine the pharmacological activity of the antagonist suramin, the most important antagonist of purinergic receptor function, as well as to demonstrate its noncompetitive inhibition and confirm a competitive mechanism between ATP and TNP-ATP in 1321N1 glial cells stably transfected with the recombinant rat P2X(2) receptor. A radioligand binding assay was employed to determine whether suramin, TNP-ATP, and ATP compete for the same binding site on the receptor. TNP-ATP displaced [alpha-32P]ATP, whereas suramin did not interfere with [alpha-32P]ATP-receptor binding. To determine the inhibition mechanism relevant for channel opening, currents obtained in fast kinetic whole-cell recording experiments, following stimulation of cells by ATP in the presence of suramin, were compared to those obtained by ATP in the presence of TNP-ATP. Supported by a mathematical model for receptor kinetics [Breitinger, H. G., Geetha, N., and Hess, G. P. (2001) Biochemistry 40, 8419-8429], the inhibition factors were plotted as functions of inhibitor or agonist concentrations. Analysis of the data indicated a competitive inhibition mechanism for TNP-ATP and a noncompetitive inhibition for suramin. Taken together, both data support a noncompetitive inhibition mechanism of the rat recombinant P2X(2) receptor by suramin, confirm the competitive inhibition by TNP-ATP, and allow the prediction of a model for P2X(2) receptor inhibition.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo 05508-900, Brazil.
authors.forename: Cleber A
authors.initials: CA
authors.lastname: Trujillo
authors.forename: Arthur A
authors.initials: AA
authors.lastname: Nery
authors.forename: Antonio Henrique B
authors.initials: AH
authors.lastname: Martins
authors.forename: Paromita
authors.initials: P
authors.lastname: Majumder
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Gonzalez
authors.forename: Henning
authors.initials: H
authors.lastname: Ulrich
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D004594:Electrophysiology; D007700:Kinetics; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology*; D014162:Transfection
chemical_list: C546403:P2rx2 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins; D013498:Suramin; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate