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.

Characterization of specific allosteric effects of the Na+ channel ß1 subunit on the Nav1.4 isoform.

The mechanism of inactivation of mammalian voltage-gated Na+ channels involves transient interactions between intracellular domains resulting in direct pore occlusion by the IFM motif and concomitant extracellular interactions with the ß1 subunit. Navß1 subunits constitute single-pass transmembrane proteins that form protein-protein associations with pore-forming α subunits to allosterically modulate the Na+ influx into the cell during the action potential of every excitable cell in vertebrates. Here, we explored the role of the intracellular IFM motif of rNav1.4 (skeletal muscle isoform of the rat Na+ channel) on the α-ß1 functional interaction and showed for the first time that the modulation of ß1 is independent of the IFM motif. We found that: (1) Nav1.4 channels that lack the IFM inactivation particle can undergo a "C-type-like inactivation" albeit in an ultraslow gating mode; (2) ß1 can significantly accelerate the inactivation of Nav1.4 channels in the absence of the IFM motif. Previously, we identified two residues (T109 and N110) on the ß1 subunit that disrupt the α-ß1 allosteric modulation. We further characterized the electrophysiological effects of the double alanine substitution of these residues demonstrating that it decelerates inactivation and recovery from inactivation, abolishes the modulation of steady-state inactivation and induces a current rundown upon repetitive stimulation, thus causing a general loss of function. Our results contribute to delineating the process of the mammalian Na+ channel inactivation. These findings may be relevant to the design of pharmacological strategies, targeting ß subunits to treat pathologies associated to Na+ current dysfunction.

Identification of Navß1 residues involved in the modulation of the sodium channel Nav1.4.

Voltage-gated sodium channels (VGSCs) are heteromeric protein complexes that initiate action potentials in excitable cells. The voltage-gated sodium channel accessory subunit, Navß1, allosterically modulates the α subunit pore structure upon binding. To date, the molecular determinants of the interface remain unknown. We made use of sequence, knowledge and structure-based methods to identify residues critical to the association of the α and ß1 Nav1.4 subunits. The Navß1 point mutant C43A disrupted the modulation of voltage dependence of activation and inactivation and delayed the peak current decay, the recovery from inactivation, and induced a use-dependent decay upon depolarisation at 1 Hz. The Navß1 mutant R89A selectively delayed channel inactivation and recovery from inactivation and had no effect on voltage dependence or repetitive depolarisations. Navß1 mutants Y32A and G33M selectively modified the half voltage of inactivation without altering the kinetics. Despite low sequence identity, highly conserved structural elements were identified. Our models were consistent with published data and may help relate pathologies associated with VGSCs to the Navß1 subunit.

A residue W756 in the P-loop segment of the sodium channel is critical for primaquine binding.

Our study on the wild-type and mutants of the voltage-dependent sodium channel in the rat skeletal muscle Na(v) 1.4 was to examine the possible binding site of primaquine PQ by using an experimental approach. We used a standard voltage-clamp in oocytes. Previously, we had demonstrated that PQ blocks the voltage-dependent sodium current in rat myocytes and that this blocking is concentration-dependent and voltage-independent. The direct-site mutagenesis in the P-loop segment W402C, W756C, W1239C, W1531A at the outer tryptophan-rich lip, and D400C, E758C, K1237C, A1529C of the DEKA locus helped us to identify residues playing a key role in aminoquinoline binding. In full agreement with our computed results, where a 1000-fold reduction of inhibition was measured, the tryptophan 756 is crucial for the reversible modulating effects of PQ. The W756C decreased the blocking effect of PQ in voltage-clamp assays. This new binding site may be important to the development of new drugs that modulate sodium inward currents.

The increase in zinc levels and upregulation of zinc transporters are mediated by nitric oxide in the cerebral cortex after transient ischemia in the rat.

The transient occlusion of cerebral arteries causes an increase in zinc levels in the brain, which is associated with a production of nitric oxide (NO). The types of zinc transporters (ZnT) involved in zinc homeostasis in the cerebral cortex after hypoxia-ischemia are not completely known. We studied the effect of the transient occlusion (10 min) of the common carotid artery (CCA) on NO-induced zinc levels, ZnT mRNA expression, and cell-death markers in the cerebral cortex-hippocampus of the rat. Nitrites, zinc, and lipoperoxidation were quantified by colorimetric methods, ZnT expression was determined by RT-PCR, caspase-3 by ELISA and immunohistochemistry, and histopathological alterations by H&E staining. After restoration of the blood flow, the basal levels of NO and zinc increased in a biphasic manner over time, but the peaks of NO levels appeared earlier (2 h and 24 h) than those of zinc (6 h and 36 h). Upregulation of ZnT1, ZnT2, and ZnT4 mRNAs was determined after 8-h postreperfusion, but ZnT3 RNA levels were unaffected. Lipoperoxidation and caspase-3 levels were also increased, and necrosis and apoptosis were present at 24 h postreperfusion. All the effects determined were prevented by l-nitro-arginine methyl ester injected 1 h before the occlusion of the CCA. Our results suggest that the upregulation of ZnT1, ZnT2, and ZnT4 was to decrease the cytosolic zinc levels caused by NO after transient occlusion of the CCA, although this was unable to lead to physiological levels of zinc and to prevent cell damage in the cerebral cortex-hippocampus of the rat.