Functional characterization of P2X<sub>3</sub>receptors fused with fluorescent proteins

Grote, Alexander; Boldogköi, Zsolt; Zimmer, Andreas; Steinhäuser, Christian; Jabs, Ronald

doi:10.1080/09687860500370638

Cited by 13 publications

(13 citation statements)

References 34 publications

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“…Therefore, the only limitation for "op" was to be high enough to allow fast activation at high agonist concentrations. Flash photolysis of 100 M caged ATP resulted in an activation with a 20% to 80% rise time of 20 ms (Grote et al, 2005). This is quite consistent with the rate constant given by Sokolova et al (2006).…”

Section: Kinetics and Affinity Of P2x 3 Receptors Explained By Model 227supporting

confidence: 73%

“…Although proper singlechannel analysis has not been performed for P2X 3 receptors, we can suppose from nonstationary fluctuation analysis data (Grote et al, 2005) that maximal open probability is not higher than ϳ0.6 to 0.8. Rate constant of desensitization from open state (od) was determined based on the results of Sokolova et al (2006), where half-times of desensitization converged to ϳ50 ms at high agonist concentrations.…”

Section: Kinetics and Affinity Of P2x 3 Receptors Explained By Model 227mentioning

confidence: 99%

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The Unusual State-Dependent Affinity of P2X₃ Receptors Can Be Explained by an Allosteric Two-Open-State Model

Karoly¹,

Mike²,

Illéš³

et al. 2007

Mol Pharmacol

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High-affinity desensitization (HAD) by nanomolar agonists was described to shape the ability of P2X 3 receptors for mediating pain sensation. These receptors are activated by micromolar ATP, but nanomolar ATP is sufficient to effectively desensitize them. The mechanism behind HAD is still obscure. It has been suggested (J Neurosci 25: 7359 -7365, 2005) that HAD can happen only if the receptor has previously been activated and desensitized by high agonist concentrations. It was not clear, however, whether the high-affinity site was different from the conventional binding site and which mechanism led to its exposure during desensitization. A subsequent article (Mol Pharmacol 70:373-382, 2006) argued that HAD could also occur without preceding desensitization, because even resting receptors expose high-affinity binding sites. To support this hypothesis, a kinetic model was proposed that could reproduce all major phenomena observed experimentally. We attempted to improve this model and used it to simulate the agonist-induced formation of the high-affinity binding site. We collected electrophysiological data using HEK 293 cells expressing human P2X 3 receptors and fitted simulated currents to experimentally acquired currents. A simple allosteric kinetic model in which only triliganded receptors could open failed to reproduce receptor behavior; introduction of an additional diliganded open state was necessary. Simulation with this model gave results that were in good agreement with experimental data. By using simulations and experiments, we analyzed the process of highaffinity binding site formation upon agonist exposure and propose an explanation, which helps to resolve the apparent conflict regarding the mechanism of HAD.Rapid desensitization (within ϳ100 ms) and very slow recovery from desensitization (requiring several minutes) are the hallmark of P2X 3 [and also of P2X 1 (Rettinger and Schmalzing, 2003)] receptors (North, 2002). In the study of this phenomenon, it has been shown that agonists compete for the same binding sites, and the recovery rate will be determined by the type of agonist occupying the binding sites of the receptors at the beginning of washout (Sokolova et al., 2004). Extremely low concentrations of agonists were found to be able to effectively inhibit agonist-evoked currents, presumably by inducing desensitization of the receptors. No detectable currents were evoked at these low concentrations; in fact, a ϳ80-to 800-fold difference was found between IC 50 and EC 50 values of the same agonist.The ability of low nanomolar agonist concentrations to induce desensitization was questioned by an elegant study of Pratt et al. (2005), where the ability of agonists to prevent recovery from desensitization was compared with their ability to induce desensitization. Intriguingly, and in contrast to previous results, no inhibition by nanomolar agonists was found without previous activation and desensitization.Dealing with a mechanism in which agonists cannot induce desensitization but, once it is at...

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Section: Kinetics and Affinity Of P2x 3 Receptors Explained By Model 227supporting

confidence: 73%

Section: Kinetics and Affinity Of P2x 3 Receptors Explained By Model 227mentioning

confidence: 99%

The Unusual State-Dependent Affinity of P2X₃ Receptors Can Be Explained by an Allosteric Two-Open-State Model

Karoly¹,

Mike²,

Illéš³

et al. 2007

Mol Pharmacol

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“…Table 3 summarizes the pharmacological profile of homomeric P2X3Rs. ATP is a full agonist for rP2X3R with estimated EC 50 values of 1.2 M (Chen et al, 1995), 2.6 M (Pratt et al, 2005), 4.1 M (Asatryan et al, 2008), and 7.3 M (Grote et al, 2005), but the precision of these estimates is limited by profound desensitization (Khmyz et al, 2008). Similar to the rP2X1R, ␣␤-meATP is a full agonist at rP2X3R, acting with a potency similar to (Pratt et al, 2005;Asatryan et al, 2008) or slightly lower than (Chen et al, 1995) that of ATP.…”

Section: Activation and Regulation Of P2xrsmentioning

confidence: 97%

“…Further studies revealed the presence of two types of receptor desensitization (Sokolova et al, 2006). As with rP2X4R , the fluorescently labeled rP2X3 subunits form fully functional channels that have biophysical and pharmacological properties highly comparable with those of the wild-type receptor (Grote et al, 2005). Table 3 summarizes the pharmacological profile of homomeric P2X3Rs.…”

Section: Activation and Regulation Of P2xrsmentioning

confidence: 99%

Activation and Regulation of Purinergic P2X Receptor Channels

et al. 2011

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“…Singlechannel measurements of rat P2X3 receptors could not resolve the individual events with 1-kHz filtering and 5-kHz sampling rates (Evans, 1996;Grote et al, 2005). We assumed that the open times of human P2X3 receptors were of the same magnitude as those from molpharm.aspetjournals.org rats.…”

Section: Methodsmentioning

confidence: 99%

Effects of Nucleotide Analogs at the P2X3 Receptor and Its Mutants Identify the Agonist Binding Pouch

Riedel

S²,

Leichsenring³

et al. 2012

Mol Pharmacol

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In this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed ATP binding site of the human P2X3 receptor on the agonistic effect of nucleotide analogs. The wild-type and mutant receptors were expressed in HEK293 cells, and the nucleotide effects were measured by means of the whole-cell patch-clamp method. Modifications in the receptor binding site changed the concentration-response relationship, the current kinetics, and the recovery from desensitization during fast, pulsed, local agonist applications. On the basis of this fact, we were able to distinguish binding from other effects, such as gating/desensitization, by using a hidden Markov model that describes the complete channel behavior with a matrix of rate constants. The binding energies of the nucleotide analogs were calculated and compared with the binding energies of ATP at both the wild-type receptor and its alanine mutants. Changes in the binding energies caused by alterations in the receptor and/or agonist structures were concluded to be attributable to the preferential binding of certain structural constituents of ATP to certain amino acid moieties of the receptor. The results were also checked for consistency with a P2X3 homology model that we developed from the known zebrafish P2X4 crystal structure in the closed state. The functional data correlated well with the predictions of the agonist dockings to the structural model.

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Functional characterization of P2X₃receptors fused with fluorescent proteins

Cited by 13 publications

References 34 publications

The Unusual State-Dependent Affinity of P2X₃ Receptors Can Be Explained by an Allosteric Two-Open-State Model

The Unusual State-Dependent Affinity of P2X₃ Receptors Can Be Explained by an Allosteric Two-Open-State Model

Activation and Regulation of Purinergic P2X Receptor Channels

Effects of Nucleotide Analogs at the P2X3 Receptor and Its Mutants Identify the Agonist Binding Pouch

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Functional characterization of P2X3receptors fused with fluorescent proteins

Cited by 13 publications

References 34 publications

The Unusual State-Dependent Affinity of P2X3 Receptors Can Be Explained by an Allosteric Two-Open-State Model

The Unusual State-Dependent Affinity of P2X3 Receptors Can Be Explained by an Allosteric Two-Open-State Model

Activation and Regulation of Purinergic P2X Receptor Channels

Effects of Nucleotide Analogs at the P2X3 Receptor and Its Mutants Identify the Agonist Binding Pouch

Contact Info

Product

Resources

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Functional characterization of P2X₃receptors fused with fluorescent proteins

The Unusual State-Dependent Affinity of P2X₃ Receptors Can Be Explained by an Allosteric Two-Open-State Model

The Unusual State-Dependent Affinity of P2X₃ Receptors Can Be Explained by an Allosteric Two-Open-State Model