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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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Application protocols used to investigate the nature of antagonism between TNP-ATP and α,β-meATP at the wild-type (wt) P2X3R and its binding site mutants.A, Steady-state application protocol for the wt P2X3R. α,β-meATP (10 µM) was superfused three times for 2 s each, with 2-s and 60-s intervals between subsequent applications, both in the absence and in the presence of increasing concentrations of TNP-ATP (0.3-30 nM; each agonist application cycle was spaced apart by 5 min). B, Dynamic antagonist application protocol. α,β-meATP (10 µM) was repetitively applied for 1 s each at an interval of 1 min. The onset and offset of the blockade by TNP-ATP (30 nM; 5 min) is shown. C, Wash-out protocol for the wt P2X3R. α,β-meATP (10 µM) application of 10-s duration was done either in the absence of TNP-ATP (30 nM) or at variable time-periods (up to 15 s, as indicated) after its wash-out; TNP-ATP was superfused for 25 s with 5 min intervals between each run. D, Concentration response-curves for the indicated mutant receptors simulated by the Markov model (lines) to fit the experimentally determined mean current amplitudes (symbols) without and with increasing concentrations of TNP-ATP (0.3 nM - 10 µM) in the superfusion medium. The F301A curve is misplaced with respect to the symbols. One possible explanation for this finding is that the simulation takes the kinetics, the association and dissociation rates and the recovery time into account and not only the amplitudes. α,β-meATP concentrations were adjusted for the requirements of every mutant. The black lines represent the experimentally measured P2X3R currents (A, C) or the lines connecting the experimentally determined mean values (B), with the grey bars as their S.E.M. The fitted currents have a red colour. Means ± S.E.M. of the data together with the generated concentration-response curves are shown in colour (D). The number of similar experiments for each group of data varied from 6-13. The thick horizontal lines above the current traces designate the duration of agonist or antagonist superfusion.
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pone-0079213-g002: Application protocols used to investigate the nature of antagonism between TNP-ATP and α,β-meATP at the wild-type (wt) P2X3R and its binding site mutants.A, Steady-state application protocol for the wt P2X3R. α,β-meATP (10 µM) was superfused three times for 2 s each, with 2-s and 60-s intervals between subsequent applications, both in the absence and in the presence of increasing concentrations of TNP-ATP (0.3-30 nM; each agonist application cycle was spaced apart by 5 min). B, Dynamic antagonist application protocol. α,β-meATP (10 µM) was repetitively applied for 1 s each at an interval of 1 min. The onset and offset of the blockade by TNP-ATP (30 nM; 5 min) is shown. C, Wash-out protocol for the wt P2X3R. α,β-meATP (10 µM) application of 10-s duration was done either in the absence of TNP-ATP (30 nM) or at variable time-periods (up to 15 s, as indicated) after its wash-out; TNP-ATP was superfused for 25 s with 5 min intervals between each run. D, Concentration response-curves for the indicated mutant receptors simulated by the Markov model (lines) to fit the experimentally determined mean current amplitudes (symbols) without and with increasing concentrations of TNP-ATP (0.3 nM - 10 µM) in the superfusion medium. The F301A curve is misplaced with respect to the symbols. One possible explanation for this finding is that the simulation takes the kinetics, the association and dissociation rates and the recovery time into account and not only the amplitudes. α,β-meATP concentrations were adjusted for the requirements of every mutant. The black lines represent the experimentally measured P2X3R currents (A, C) or the lines connecting the experimentally determined mean values (B), with the grey bars as their S.E.M. The fitted currents have a red colour. Means ± S.E.M. of the data together with the generated concentration-response curves are shown in colour (D). The number of similar experiments for each group of data varied from 6-13. The thick horizontal lines above the current traces designate the duration of agonist or antagonist superfusion.

Mentions: (1) Steadystateprotocol (e.g. Figure 2A). In this protocol, we combined the construction of a concentration-response curve for the antagonist and the measurement of receptor kinetics (recovery from desensitization; [16]) by repetitively applying the agonist. In every run with increasing antagonist concentrations, the same concentration of the agonist was applied (2-s duration), 28 s, 32 s and 94 s after starting antagonist superfusion. After 5 minutes, which is sufficient for P2X3R to recover from desensitization, the next run with an increasing antagonist concentration was started. This protocol provides information about the concentration-inhibition relationship for antagonists, but gives no information about the kinetics of their receptor association and -dissociation.


Agonist antagonist interactions at the rapidly desensitizing P2X3 receptor.

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

Application protocols used to investigate the nature of antagonism between TNP-ATP and α,β-meATP at the wild-type (wt) P2X3R and its binding site mutants.A, Steady-state application protocol for the wt P2X3R. α,β-meATP (10 µM) was superfused three times for 2 s each, with 2-s and 60-s intervals between subsequent applications, both in the absence and in the presence of increasing concentrations of TNP-ATP (0.3-30 nM; each agonist application cycle was spaced apart by 5 min). B, Dynamic antagonist application protocol. α,β-meATP (10 µM) was repetitively applied for 1 s each at an interval of 1 min. The onset and offset of the blockade by TNP-ATP (30 nM; 5 min) is shown. C, Wash-out protocol for the wt P2X3R. α,β-meATP (10 µM) application of 10-s duration was done either in the absence of TNP-ATP (30 nM) or at variable time-periods (up to 15 s, as indicated) after its wash-out; TNP-ATP was superfused for 25 s with 5 min intervals between each run. D, Concentration response-curves for the indicated mutant receptors simulated by the Markov model (lines) to fit the experimentally determined mean current amplitudes (symbols) without and with increasing concentrations of TNP-ATP (0.3 nM - 10 µM) in the superfusion medium. The F301A curve is misplaced with respect to the symbols. One possible explanation for this finding is that the simulation takes the kinetics, the association and dissociation rates and the recovery time into account and not only the amplitudes. α,β-meATP concentrations were adjusted for the requirements of every mutant. The black lines represent the experimentally measured P2X3R currents (A, C) or the lines connecting the experimentally determined mean values (B), with the grey bars as their S.E.M. The fitted currents have a red colour. Means ± S.E.M. of the data together with the generated concentration-response curves are shown in colour (D). The number of similar experiments for each group of data varied from 6-13. The thick horizontal lines above the current traces designate the duration of agonist or antagonist superfusion.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815160&req=5

pone-0079213-g002: Application protocols used to investigate the nature of antagonism between TNP-ATP and α,β-meATP at the wild-type (wt) P2X3R and its binding site mutants.A, Steady-state application protocol for the wt P2X3R. α,β-meATP (10 µM) was superfused three times for 2 s each, with 2-s and 60-s intervals between subsequent applications, both in the absence and in the presence of increasing concentrations of TNP-ATP (0.3-30 nM; each agonist application cycle was spaced apart by 5 min). B, Dynamic antagonist application protocol. α,β-meATP (10 µM) was repetitively applied for 1 s each at an interval of 1 min. The onset and offset of the blockade by TNP-ATP (30 nM; 5 min) is shown. C, Wash-out protocol for the wt P2X3R. α,β-meATP (10 µM) application of 10-s duration was done either in the absence of TNP-ATP (30 nM) or at variable time-periods (up to 15 s, as indicated) after its wash-out; TNP-ATP was superfused for 25 s with 5 min intervals between each run. D, Concentration response-curves for the indicated mutant receptors simulated by the Markov model (lines) to fit the experimentally determined mean current amplitudes (symbols) without and with increasing concentrations of TNP-ATP (0.3 nM - 10 µM) in the superfusion medium. The F301A curve is misplaced with respect to the symbols. One possible explanation for this finding is that the simulation takes the kinetics, the association and dissociation rates and the recovery time into account and not only the amplitudes. α,β-meATP concentrations were adjusted for the requirements of every mutant. The black lines represent the experimentally measured P2X3R currents (A, C) or the lines connecting the experimentally determined mean values (B), with the grey bars as their S.E.M. The fitted currents have a red colour. Means ± S.E.M. of the data together with the generated concentration-response curves are shown in colour (D). The number of similar experiments for each group of data varied from 6-13. The thick horizontal lines above the current traces designate the duration of agonist or antagonist superfusion.
Mentions: (1) Steadystateprotocol (e.g. Figure 2A). In this protocol, we combined the construction of a concentration-response curve for the antagonist and the measurement of receptor kinetics (recovery from desensitization; [16]) by repetitively applying the agonist. In every run with increasing antagonist concentrations, the same concentration of the agonist was applied (2-s duration), 28 s, 32 s and 94 s after starting antagonist superfusion. After 5 minutes, which is sufficient for P2X3R to recover from desensitization, the next run with an increasing antagonist concentration was started. This protocol provides information about the concentration-inhibition relationship for antagonists, but gives no information about the kinetics of their receptor association and -dissociation.

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