Inhibition of the Human Neuronal Sodium Channel Nav1.9 by 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 (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.

Mefloquine inhibits voltage dependent Nav1.4 channel by overlapping the local anaesthetic binding site.

Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na+ channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na+ channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Nav1.4 currents (IC50 =60µM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the ß1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Nav1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na+ channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources.

Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 ß1).

The molecular structure modeling of the ß1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the ß1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the ß1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of "all ß proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another "all ß protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect). This finding coincides with our electrophysiological data (disrupted ß1-like voltage dependence) and it is safe to utter that the Nav1.4 α/ß1 interface is likely to be of reversible nature.