title: Kinetics of antagonist actions at rat P2X2/3 heteromeric receptors.
abstract: 1. Currents through heteromeric P2X(2/3) receptors were evoked by applying alpha,beta-methylene-ATP to human embryonic kidney cells transfected with cDNAs encoding the P2X(2) and P2X(3) subunits. The concentration of alpha,beta-methylene-ATP were < or =30 microM because higher concentrations can activate homomeric P2X(2) receptors. The kinetics of action of three structurally unrelated antagonists were studied; these were 2', 3'-O-(2,4,6,trinitrophenyl)-ATP (TNP-ATP), pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. The association and dissociation rate constants were determined by pre-applying the antagonist for various periods prior to the co-application of agonist and antagonist, or by changing the solution from one containing only the agonist to one containing both agonist and antagonist. The high affinity of TNP-ATP for the P2X(2/3) receptor (K(D) approximately 2 nM) results from fast binding (k(+1) approximately 100 microM(-1) s(-1)) rather than slow unbinding (k(-1) approximately 0.3 s(-1)). For suramin (K(D) approximately 1 microM) the association rate constant ( approximately 1 microM(-1) s(-1)) was 100 times slower than that of TNP-ATP but the dissociation rate constant was similar (k(-1) approximately 1 s(-1)). PPADS (K(D) approximately 0.1 microM) associated and dissociated some 100 - 10,000 times more slowly than the other antagonists.
authors.affiliation: Institute of Molecular Physiology, University of Sheffield, Alfred Denny Building, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Valeria
authors.initials: V
authors.lastname: Spelta
authors.forename: Lin-Hua
authors.initials: LH
authors.lastname: Jiang
authors.forename: Annmarie
authors.initials: A
authors.lastname: Surprenant
authors.forename: R Alan
authors.initials: RA
authors.lastname: North
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D010975:Platelet Aggregation Inhibitors / Q000493:pharmacokinetics; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D013498:Suramin / Q000493:pharmacokinetics*; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; C546401:P2RX2 protein, human; C546407:P2RX3 protein, human; C546403:P2rx2 protein, rat; C546410:P2rx3 protein, rat; D010975:Platelet Aggregation Inhibitors; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D013498:Suramin; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate