Received for publication June 12, 2007.
Revised September 20, 2007.
Accepted for publication October 9, 2007.

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 P2X₃ 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, that HAD can only happen 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 paper 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, which 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 P2X₃ 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 which were in good agreement with experimental data. By using simulations and experiments we analyzed the process of high affinity binding site formation upon agonist exposure, and propose an explanation, which helps to resolve the apparent conflict regarding the mechanism of HAD.

Key words: Purinergic, Single channel kinetics