Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models

The P2X4 receptor (P2X4R) is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM), a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4R. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.Author summary: Ligand-gated ion channels play a crucial role in controlling many physiological and pathophysiological processes. Deciphering the gating kinetics of these channels is thus fundamental to understanding how these processes work. ATP-gated purinergic P2X receptors (P2XRs) are prototypic examples of such channels. They are ubiquitously expressed and play roles in numerous cellular processes, including neurotransmission, inflammation, and chronic pain. Seven P2X subunits, named P2X1 through P2X7, and several splice forms of these subunits have been identified in mammal. The receptors are organized as homo- or heterotrimers, each possessing three ATP-binding sites that, when occupied, lead to receptor activation and channel opening. The P2XRs are non-selective cation channels and the gating properties differ between the various receptors. Previously, we have used biophysical and mathematical modeling approaches to decipher the kinetics of homomeric P2X2aR, P2X2bR, P2X4R, and P2X7R. Here we extended our work on P2X4R gating. We developed two mathematical models that could capture the various patterns of ionic currents recorded experimentally and explain the particularly complex kinetics of the receptor during orthosteric activation and allosteric modulation. This was achieved by designing a computationally efficient, inference-based fitting algorithm that allowed for parameter optimization and model comparisons.