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Agonist antagonist interactions at the rapidly desensitizing P2X3 receptor.

Helms N, Kowalski M, Illes P, Riedel T - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Rudolf Boehm Institute for Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.

ABSTRACT
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.

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Illustration of the influence of P2X3R desensitization on the Schild-analysis of agonist effects.Concentration-response curves of α,β-meATP in the presence and absence of increasing A317491 concentrations were simulated by the wt P2X3 model (A) and with the same model without desensitization (B). The symbols represent the simulated data points and the lines the corresponding hill fits. A, High agonist concentrations did not induce maximal current amplitudes in the presence of the antagonist. This is due to the fast receptor desensitization which suppresses the current before equilibrium between the agonist and its antagonist is reached at the binding site. The decreased maxima and the non-parallel displacement of the agonist concentration-response curves suggest non-competitive antagonism. B, After setting the desensitization rates (d1-d4) to zero, the competitive character of the model is unmasked. C, The Schild-plot (inset) shows the expected straight line. I (a.u.), current in arbitrary units.
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pone-0079213-g005: Illustration of the influence of P2X3R desensitization on the Schild-analysis of agonist effects.Concentration-response curves of α,β-meATP in the presence and absence of increasing A317491 concentrations were simulated by the wt P2X3 model (A) and with the same model without desensitization (B). The symbols represent the simulated data points and the lines the corresponding hill fits. A, High agonist concentrations did not induce maximal current amplitudes in the presence of the antagonist. This is due to the fast receptor desensitization which suppresses the current before equilibrium between the agonist and its antagonist is reached at the binding site. The decreased maxima and the non-parallel displacement of the agonist concentration-response curves suggest non-competitive antagonism. B, After setting the desensitization rates (d1-d4) to zero, the competitive character of the model is unmasked. C, The Schild-plot (inset) shows the expected straight line. I (a.u.), current in arbitrary units.

Mentions: It is difficult to compare results obtained by different research groups with respect to P2X3 antagonists, because they have not been systematically compared in the same preparation and because inadequate experimental protocols, e.g. preincubation times with antagonists not sufficient to reach steady-state conditions, were used [15]. Furthermore, it is not possible to decide by a classic analysis of agonist-antagonist interaction (e.g. Schild plot) whether α,β-meATP and its antagonists interact in a competitive or non-competitive manner at the rapidly desensitizing P2X3R (for P2X1 see 23). The interaction between an agonist and its antagonist is not a simple displacement under equilibrium conditions, but it is complicated by desensitization, because not only the peak current amplitude, but also the current kinetics are altered in the presence of the antagonist. This results in a non-parallel shift of the concentration-response curves for α,β-meATP, and a marked depression of the peak current amplitude (Figure 5A) and may lead to the false conclusion that TNP-ATP acts in a non-competitive manner [19]. By contrast, simulation of the curves without desensitization (by setting the desensitization rates to zero) results in parallel shifts to the right with no change in the respective maxima, allowing the proper determination of the pA2 value (Figure 5B)


Agonist antagonist interactions at the rapidly desensitizing P2X3 receptor.

Helms N, Kowalski M, Illes P, Riedel T - PLoS ONE (2013)

Illustration of the influence of P2X3R desensitization on the Schild-analysis of agonist effects.Concentration-response curves of α,β-meATP in the presence and absence of increasing A317491 concentrations were simulated by the wt P2X3 model (A) and with the same model without desensitization (B). The symbols represent the simulated data points and the lines the corresponding hill fits. A, High agonist concentrations did not induce maximal current amplitudes in the presence of the antagonist. This is due to the fast receptor desensitization which suppresses the current before equilibrium between the agonist and its antagonist is reached at the binding site. The decreased maxima and the non-parallel displacement of the agonist concentration-response curves suggest non-competitive antagonism. B, After setting the desensitization rates (d1-d4) to zero, the competitive character of the model is unmasked. C, The Schild-plot (inset) shows the expected straight line. I (a.u.), current in arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815160&req=5

pone-0079213-g005: Illustration of the influence of P2X3R desensitization on the Schild-analysis of agonist effects.Concentration-response curves of α,β-meATP in the presence and absence of increasing A317491 concentrations were simulated by the wt P2X3 model (A) and with the same model without desensitization (B). The symbols represent the simulated data points and the lines the corresponding hill fits. A, High agonist concentrations did not induce maximal current amplitudes in the presence of the antagonist. This is due to the fast receptor desensitization which suppresses the current before equilibrium between the agonist and its antagonist is reached at the binding site. The decreased maxima and the non-parallel displacement of the agonist concentration-response curves suggest non-competitive antagonism. B, After setting the desensitization rates (d1-d4) to zero, the competitive character of the model is unmasked. C, The Schild-plot (inset) shows the expected straight line. I (a.u.), current in arbitrary units.
Mentions: It is difficult to compare results obtained by different research groups with respect to P2X3 antagonists, because they have not been systematically compared in the same preparation and because inadequate experimental protocols, e.g. preincubation times with antagonists not sufficient to reach steady-state conditions, were used [15]. Furthermore, it is not possible to decide by a classic analysis of agonist-antagonist interaction (e.g. Schild plot) whether α,β-meATP and its antagonists interact in a competitive or non-competitive manner at the rapidly desensitizing P2X3R (for P2X1 see 23). The interaction between an agonist and its antagonist is not a simple displacement under equilibrium conditions, but it is complicated by desensitization, because not only the peak current amplitude, but also the current kinetics are altered in the presence of the antagonist. This results in a non-parallel shift of the concentration-response curves for α,β-meATP, and a marked depression of the peak current amplitude (Figure 5A) and may lead to the false conclusion that TNP-ATP acts in a non-competitive manner [19]. By contrast, simulation of the curves without desensitization (by setting the desensitization rates to zero) results in parallel shifts to the right with no change in the respective maxima, allowing the proper determination of the pA2 value (Figure 5B)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Rudolf Boehm Institute for Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus