Abstract：
Cannabinoids regulate analgesia, which has aroused much interest in identifying new pharmacological therapies in the management of refractory pain. Voltage-gated Na+ channels (Navs) play an important role in inflammatory and neuropathic pain. In particular, Nav1.9 is involved in nociception and the understanding of its pharmacology has lagged behind because it is difficult to express in heterologous systems. Here, we utilized the chimeric channel hNav1.9_C4, that comprises the extracellular and transmembrane domains of hNav1.9, co-expressed with the ß1 subunit on CHO-K1 cells to characterize the electrophysiological effects of ACEA, a synthetic surrogate of the endogenous cannabinoid anandamide. ACEA induced a tonic block, decelerated the fast inactivation, markedly shifted steady-state inactivation in the hyperpolarized direction, decreasing the window current and showed use-dependent block, with a high affinity for the inactivated state (ki = 0.84 µM). Thus, we argue that ACEA possess a local anaesthetic-like profile. To provide a mechanistic understanding of its mode of action at the molecular level, we combined induced fit docking with Monte Carlo simulations and electrostatic complementarity. In agreement with the experimental evidence, our computer simulations revealed that ACEA binds Tyr1599 of the local anaesthetics binding site of the hNav1.9, contacting residues that bind cannabinol (CBD) in the NavMs channel. ACEA adopted a conformation remarkably similar to the crystallographic conformation of anandamide on a non-homologous protein, obstructing the Na+ permeation pathway below the selectivity filter to occupy a highly conserved binding pocket at the intracellular side. These results describe a mechanism of action, possibly involved in cannabinoid analgesia.
摘要：
大麻素调节镇痛，这引起了人们对确定难治性疼痛的新药物治疗方法的极大兴趣。电压门控Na+通道(Navs)在炎性和神经性疼痛中起重要作用。特别是,Nav1.9涉及伤害感受，对其药理学的理解滞后，因为它很难在异源系统中表达。这里,我们利用嵌合通道hNav1.9_C4，它包含hNav1.9的细胞外和跨膜结构域，与CHO-K1细胞上的β1亚基共表达，以表征ACEA的电生理效应，内源性大麻素anandamide的合成替代品。ACEA诱发了强直阻滞，减速快速失活，在超极化方向上显著移动的稳态失活，降低窗口电流并显示依赖使用的块，对失活状态具有高亲和力（ki=0.84µM）。因此，我们认为ACEA具有类似局部麻醉的特征。为了在分子水平上提供对其作用方式的机械理解，我们将诱导拟合对接与蒙特卡罗模拟和静电互补相结合。与实验证据一致，我们的计算机模拟显示,ACEA结合hNav1.9局部麻醉剂结合位点的Tyr1599,接触NavMs通道中结合大麻酚(CBD)的残基.ACEA在非同源蛋白上采用了与anandamide的晶体学构象非常相似的构象，阻碍选择性过滤器下方的Na+渗透途径以占据细胞内侧的高度保守的结合袋。这些结果描述了一种作用机制，可能涉及大麻素镇痛。
