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

Abstract

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.

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).…”

Purinergic P2X Receptors: Structural and Functional Features Depicted by X-Ray and Molecular Modelling Studies

“…Both studies required the molecular models to enable experimental design. Riedel et al made a model of rP2X3 using Modeller, and then used Autodock Vina to dock ATP and its analogues into the binding site [109]. On superficial inspection ATP does not appear to adopt the U-shaped conformation seen in the crystal structure of zfP2X4.1 with ATP bound [16], but as previously observed, there is some evidence that ATP may be able to bind to P2X receptors in more than one conformation [11].…”

Agonist Antagonist Interactions at the Rapidly Desensitizing P2X3 Receptor

“…On the basis of a recently published Markov model, which describes the behaviour of P2X3R-channels during agonist binding [16], we created an extended model also accounting for antagonist actions. In the present extended model, we supposed that the binding of a competitive antagonist is just an alternative step to the binding of an agonist, and has no further consequences for the receptor, except to prevent agonist binding.…”

“…We extended a previously developed Markov model for agonist binding [16] with further parameters to model also antagonist binding. Eventually, a minimum number of two parameters (the association and dissociation rates of antagonists) were sufficient to simulate a variety of experimental conditions, such as the concentration-dependence of inhibition and the wash-in and wash-out kinetics.…”