Understanding the Kinetics of ATP-Activated P2X2A and P2X2B Receptor Channels using a Markov State Model

Abstract

ATP-gated P2X2 receptors exhibit two opposite activation-dependent changes during sustained agonist application, pore dilation and pore closing (desensitization), through a process that is incompletely understood. To address this issue and to clarify the roles of Ca2+ and the C-terminal domain in gating, we combined biophysical and mathematical approaches using the full size receptor (labeled P2X2aR) and the splice form missing 69 residues in the C-terminal domain (labeled the P2X2bR). Both forms of the receptor developed conductivity for large organic cations within 2-6 s of ATP application and desensitized in a Ca2+ influx-dependent manner, whereas P2X2bR also desensitized in a Ca2+ influx-independent manner. In whole-cell recording with broken membranes, we also observed use-dependent facilitation of desensitization, reflecting the altered Ca2+ handling by cells. Such behavior was accounted for by a Markov state kinetic model with 12 states describing the ATP binding/unbinding and activation/desensitization. The model assumes that naive receptors open when two ATP molecules bind and slowly dilates to a higher conductance state when a third ATP binds, generating a shift to less negative reversal potential observed experimentally in organic cation-containing medium. The use-dependent desensitization is modeled by a Ca2+-dependent toggle switch, whereas the P2X2bR model also exhibits fast Ca2+-independent desensitization. The model is extended to include memory to previous stimulations that not only explained the decrease in the slope of the IV-curves during −80 to +80 voltage ramps delivered twice per second, but also captured the effect of ATP stimulation when cells were held at positive holding potential.