Eduardo M. Salinas-Stefanon

Inhibition of cardiac sodium currents by toluene exposure

Toluene is an industrial solvent widely used as a drug of abuse, which can produce sudden sniffing death due to cardiac arrhythmias. In this paper, we tested the hypothesis that toluene inhibits cardiac sodium channels in Xenopus laevis oocytes transfected with Nav1.5 cDNA and in isolated rat ventricular myocytes. In oocytes, toluene inhibited sodium currents (INa+) in a concentration‐dependent manner, with an IC50 of 274 μM (confidence limits: 141–407μM). The inhibition was complete, voltage‐independent, and slowly reversible. Toluene had no effect on: (i) the shape of the I–V curves; (ii) the reversal potential of Na+; and (iii) the steady‐state inactivation. The slow recovery time constant from inactivation of INa+ decreased with toluene exposure, while the fast recovery time constant remained unchanged. Block of INa+ by toluene was use‐ and frequency‐dependent. In rat cardiac myocytes, 300 μM toluene inhibited the sodium current (INa+) by 62%; this inhibition was voltage independent. These results suggest that toluene binds to cardiac Na+ channels in the open state and unbinds either when channels move between inactivated states or from an inactivated to a closed state. The use‐ and frequency‐dependent block of INa+ by toluene might be responsible, at least in part, for its arrhythmogenic effect.

Inhibition of cardiac Na+ current by primaquine

The electrophysiological effects of the anti‐malarial drug primaquine on cardiac Na+ channels were examined in isolated rat ventricular muscle and myocytes. In isolated ventricular muscle, primaquine produced a dose‐dependent and reversible depression of dV/dt during the upstroke of the action potential. In ventricular myocytes, primaquine blocked INa+ in a dose‐dependent manner, with a Kd of 8.2 μM. Primaquine (i) increased the time to peak current, (ii) depressed the slow time constant of INa+ inactivation, and (iii) slowed the fast component for recovery of INa+ from inactivation. Primaquine had no effect on: (i) the shape of the I – V curve, (ii) the reversal potential for Na+, (iii) the steady‐state inactivation and gNa+ curves, (iv) the fast time constant of inactivation of INa+, and (v) the slow component of recovery from inactivation. Block of INa+ by primaquine was use‐dependent. Data obtained using a post‐rest stimulation protocol suggested that there was no closed channel block of Na+ channels by primaquine. These results suggest that primaquine blocks cardiac Na+ channels by binding to open channels and unbinding either when channels move between inactivated states or from an inactivated state to a closed state. Cardiotoxicity observed in patients undergoing malaria therapy with aminoquinolines may therefore be due to block of Na+ channels, with subsequent disturbances of impulse conductance and contractility.

In silico modeling of toluene binding site in the pore of voltage-gate sodium channel

Toluene is a commonly used organic solvent in commercial products and is sometimes abused as an inhalative hallucinogen, causing arrythmogenic toxicity. At a molecular level we investigated whether a hypothetical interaction model could be devised for the reported myo- and cardiotoxic effects of toluene. Three lines of computed evidence support our hypothesis on the interaction mechanism: (i) Toluene binds at the local anesthetic binding site (LABS), on the wild type (WT) but not on its F1579A mutation, confi rming our experimental fi ndings that it inhibits only the WT of skeletal muscle or cardiac isoforms (Na v 1.4 or 1.5). (ii) Typically for small alkylaryl moiety, multiple binding modes were detected during docking. Toluene is trapped in the tryptophane-rich area at the extracellular vestibule by hydrophobic interaction, mainly π-π stacking, or bound to the LABS with equal binding strength and number of solved poses, mostly by edge-to-face contacts. (iii) The computed loss of toluene binding at the LABS on the mutant model parallels clearly the observed loss of toluene effects on Na v 1.4. Moreover, we inspected the complete primary sequences with the omitted loops in the 3D models to identify the possible interacting amino acids among the 16% nonidentical ones, and thus confi rmed the observed toxicity effects. + channel, Na v 1.4 isoform, Na v 1.5 isoform, in-silico simulation, cardiotoxicity

Identification of Navβ1 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, Navβ1, 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 β1 Nav1.4 subunits. The Navβ1 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 Navβ1 mutant R89A selectively delayed channel inactivation and recovery from inactivation and had no effect on voltage dependence or repetitive depolarisations. Navβ1 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 Navβ1 subunit.

An in silico approach to primaquine binding to Trp756 in the external vestibule of sodium channel Na v 1.4

The aim of our computed study was to examine the possible binding site of primaquine (PQ) using a combined homology protein modeling, automated docking, and experimental approach. The target models of wild type and mutant types of the voltage- dependent sodium channel in rat skeletal muscle (rNa v 1.4) were based on previous work by other researchers. Docking was carried out on the P-loop in the structural model of the rNa v 1.4 channel, in the open state configuration, to identify those amino acidic residues important for PQ binding. The three-dimensional models of the P-loop segment of wild types and mutant types (W402, W756C, W1239C, and W1531A at the outer tryptophan-rich lip, as well as D400C, E755C, K1237C, and A1529C of the DEKA motif) helped us to identify residues playing key roles in aminoquinoline binding. In good agreement with experimental results, a 1000-fold inhibition loss was observed. Tryptophan 756 is crucial for the reversible blocking effects of PQ. As a result, mutant-type W756C abolished the blocking effect of PQ in voltage-clamp

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

ABSTRACT 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 =60 &mgr;M), 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 &bgr;1 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 &pgr;‐&pgr; 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.

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.

Characterization of specific allosteric effects of the Na+ channel β1 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 β1 subunit. Navβ1 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-β1 functional interaction and showed for the first time that the modulation of β1 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) β1 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 β1 subunit that disrupt the α-β1 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 β subunits to treat pathologies associated to Na+ current dysfunction.

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 β1)

The molecular structure modeling of the β1 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 β1 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 β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of “all β 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 β 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 β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.

Chlorthalidone inhibits the KvLQT1 potassium current in guinea-pig ventricular myocytes and oocytes from Xenopus laevis

Chlorthalidone is used for the treatment of hypertension as it produces a lengthening of the cardiac action potential. However, there is no experimental evidence that chlorthalidone has electrophysiological effects on the potassium currents involved in cardiac repolarization.