Inhibition of the human neuronal sodium channel Nav1.9 by ACEA (arachidonyl-2-chloroethylamide), an analogue of anandamide in a hNav1.9/rNav1.4 chimera, an experimental and in silico study

Cannabinoids regulate analgesia, which has aroused much interest in identifying new pharmacological therapies in the management of refractory pain. Voltage-gated Na+ channels (Na_vs) play an important role in inflammatory and neuropathic pain. In particular, Na_v1.9 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 hNa_v1.9_C4, that comprises the extracellular and transmembrane domains of hNa_v1.9, 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 (k_i = 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 hNa_v1.9, contacting residues that bind cannabinol (CBD) in the Na_vMs 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.