Kinetics of antagonist actions at rat P2X 2/3 heteromeric receptors

Currents through heteromeric P2X 2/3 receptors were evoked by applying α,β‐methylene‐ATP to human embryonic kidney cells transfected with cDNAs encoding the P2X 2 and P2X 3 subunits. The concentration of α,β‐methylene‐ATP were 30 μ M 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 ∼2 n M ) results from fast binding ( k +1 ∼100 μ M −1  s −1 ) rather than slow unbinding ( k −1 ∼0.3 s −1 ). For suramin ( K D ∼1 μ M ) the association rate constant (∼1 μ M −1  s −1 ) was 100 times slower than that of TNP‐ATP but the dissociation rate constant was similar ( k −1  ∼1 s −1 ). PPADS ( K D ∼0.1 μ M ) associated and dissociated some 100 – 10,000 times more slowly than the other antagonists.British Journal of Pharmacology (2002) 135 , 1524–1530; doi: 10.1038/sj.bjp.0704591