Agonist antagonist interactions at the rapidly desensitizing P2X3 receptor.
P2X3 receptors (P2XRs), as members of the purine receptor family, are deeply involved in chronic pain sensation and therefore, specific, competitive antagonists are of great interest for perspective pain management. Heretofore, Schild plot analysis has been commonly used for studying the interaction of competitive antagonists and the corresponding receptor. Unfortunately, the steady-state between antagonist and agonist, as a precondition for this kind of analysis, cannot be reached at fast desensitizing receptors like P2X3R making Schild plot analysis inappropriate. The aim of this study was to establish a new method to analyze the interaction of antagonists with their binding sites at the rapidly desensitizing human P2X3R. The patch-clamp technique was used to investigate the structurally divergent, preferential antagonists A317491, TNP-ATP and PPADS. The P2X1,3-selective α,β-methylene ATP (α,β-meATP) was used as an agonist to induce current responses at the wild-type (wt) P2X3R and several agonist binding site mutants. Afterwards a Markov model combining sequential transitions of the receptor from the closed to the open and desensitized mode in the presence or absence of associated antagonist molecules was developed according to the measured data. The P2X3R-induced currents could be fitted correctly with the help of this Markov model allowing identification of amino acids within the binding site which are important for antagonist binding. In conclusion, Markov models are suitable to simulate agonist antagonist interactions at fast desensitizing receptors such as the P2X3R. Among the antagonists investigated, TNP-ATP and A317491 acted in a competitive manner, while PPADS was identified as a (pseudo)irreversible blocker.
Rudolf Boehm Institute for Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.
Nick
N
Helms
Maria
M
Kowalski
Peter
P
Illes
Thomas
T
Riedel
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D004347:Drug Interactions; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008390:Markov Chains; D008564:Membrane Potentials / Q000187:drug effects; D008954:Models, Biological; D009154:Mutation; D018408:Patch-Clamp Techniques; D010636:Phenols / Q000378:metabolism / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D058910:Purinergic P2 Receptor Agonists / Q000378:metabolism* / Q000494:pharmacology; D058919:Purinergic P2 Receptor Antagonists / Q000378:metabolism* / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D058477:Receptors, Purinergic P2X3 / Q000235:genetics / Q000378:metabolism*
C470346:A-317491; D010636:Phenols; D011083:Polycyclic Compounds; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D058477:Receptors, Purinergic P2X3; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate