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

Abstract

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...

Lipid Rafts Control P2X3 Receptor Distribution and Function in Trigeminal Sensory Neurons of a Transgenic Migraine Mouse Model

“…In the case of P2X3 receptors, kinetic modelling is advantageous to explore certain receptor operation steps which develop very rapidly and are readily obscured by intervening desensitization, making quite difficult to extract detailed information from single channel studies [33,34]. …”

Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors

“…of P2X3 encoding cDNA (P2X2:P2X3 cDNA ratio 0.5) (Wilkinson et al, 2006;Hausmann et al, 2012). From electrophysiological analysis and mathematical modelling of the activation and deactivation kinetics of the P2X3 receptors it was derived that occupation of already a minimum of two of the available three binding sites is sufficient to open the P2X3 receptor-channel (Karoly et al, 2008;Riedel et al, 2012). A similar conclusion was reached for P2X2/3 and P2X2/6 heteromeric receptors by ATP-binding site mutagenesis and functional analysis (Hausmann et al, 2012) and the P2X2 receptor by using concatenated P2X2 receptors (Stelmashenko et al, 2012).…”

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

“…The Markov model for P2X3 used in this study ( Fig. 2C) (for detailed description, see Results) was based on proven models (Sokolova et al, 2006;Karoly et al, 2008). The fixed factors that ensure microscopic reversibility were replaced by the more-flexible loop-balance function provided by QuB software (http://www.qub.buffalo.edu) (Milescu et al, 2005).…”

“…The kinetic model used by us was based on a refinement of the original cyclic model for P2X3 operation described by Sokolova et al (2006). We added an additional step to the model, assuming that both diligand and triliganded receptors could open upon agonist exposure (Karoly et al, 2008). This correction resulted in better fits of the P2X3 current traces.…”

“…The original model assumed that each P2X subunit contains one individual binding site for ATP (Vial et al, 2004), but it soon became clear that binding probably occurs at the interface between two neighboring subunits (Wilkinson et al, 2006;Marquez-Klaka et al, 2009). Earlier electrophysiological studies (Bean, 1990;Ding and Sachs, 1999) and molecular simulation of the agonist-induced currents (Sokolova et al, 2006;Karoly et al, 2008) both suggested that at least two agonist molecules are necessary for receptor activation. The published crystal structure of the zebrafish (zf) P2X4 receptor at a resolution of 3.1 Å fully confirmed both the trimeric architecture of the P2X receptor family and the location of the agonist binding pouch between two subunits in the extracellular domain Browne et al, 2010;Young, 2010).…”