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.P2X4受体（P2X4 receptor, P2X4R）是一类嘌呤能离子通道家族成员，可被细胞外ATP激活，其包含三个正构结合位点，且可被广谱抗寄生虫药剂伊维菌素（ivermectin, IVM）进行别构调控。伊维菌素处理可增强ATP激活P2X4R的效能，延缓持续ATP暴露时的受体脱敏过程与ATP洗脱后的受体失活过程，同时使受体通道对大型有机阳离子N-甲基-D-葡萄糖阳离子（NMDG+）具有通透性。此前，本研究团队已基于单个伊维菌素结合位点构建了马尔可夫模型（Markov model），可部分描述伊维菌素对大鼠P2X4R的调控效应。本文报道了两种全新的、均包含三个伊维菌素结合位点的模型：其中较为简易的单层模型可复现诸多已观测到的诱发电流时间序列，但无法准确捕捉激活、脱敏与失活的短时尺度特征；更为复杂的双层模型则可复现伊维菌素处理后观测到的脱敏瞬时变化、低浓度伊维菌素下ATP诱导电流振幅的显著提升，以及单通道电导的小幅升高。此外，该双层模型还提示该受体可处于不响应ATP的深度失活状态，且三个伊维菌素结合位点均可改变受体的脱敏速率。综上，本研究对P2X4受体的动力学特性进行了详尽分析，并阐明了调控其通道门控的正构与别构机制。