Sodium Channel Pore Research Articles

Binding of diverse inhibitors in the sodium channel pore

Binding of diverse inhibitors in the sodium channel pore

Size and drug accessibility of fenestrations in voltage-gated sodium channel subtypes

Voltage-gated sodium channels (Nav) are integral cell membrane proteins, responsible for electrical signalling in the heart, brain and muscle. Nine different subtypes of these channels are found throughout the body and are inhibited by numerous anti-arrhythmic, local anaesthetic and anti-epileptic medications. These inhibitors have been found to access their binding site in the sodium channel pore cavity via the lateral fenestrations, which occur between adjacent pore domains. The differential accessibility via these fenestrations is a potential avenue for selective drugs to target different Nav subtypes, however, the dimensions of the four non-identical fenestrations and particularly the structural differences between the nine subtypes is yet to be determined.

Lacosamide Inhibition of NaV1.7 Channels Depends on its Interaction With the Voltage Sensor Domain and the Channel Pore.

Lacosamide, developed as an anti-epileptic drug, has been used for the treatment of pain. Unlike typical anticonvulsants and local anesthetics which enhance fast-inactivation and bind within the pore of sodium channels, lacosamide enhances slow-inactivation of these channels, suggesting different binding mechanisms and mode of action. It has been reported that lacosamide’s effect on NaV1.5 is sensitive to a mutation in the local anesthetic binding site, and that it binds with slow kinetics to the fast-inactivated state of NaV1.7. We recently showed that the NaV1.7-W1538R mutation in the voltage-sensing domain 4 completely abolishes NaV1.7 inhibition by clinically-achievable concentration of lacosamide. Our molecular docking analysis suggests a role for W1538 and pore residues as high affinity binding sites for lacosamide. Aryl sulfonamide sodium channel blockers are also sensitive to substitutions of the W1538 residue but not of pore residues. To elucidate the mechanism by which lacosamide exerts its effects, we used voltage-clamp recordings and show that lacosamide requires an intact local anesthetic binding site to inhibit NaV1.7 channels. Additionally, the W1538R mutation does not abrogate local anesthetic lidocaine-induced blockade. We also show that the naturally occurring arginine in NaV1.3 (NaV1.3-R1560), which corresponds to NaV1.7-W1538R, is not sufficient to explain the resistance of NaV1.3 to clinically-relevant concentrations of lacosamide. However, the NaV1.7-W1538R mutation conferred sensitivity to the NaV1.3-selective aryl-sulfonamide blocker ICA-121431. Together, the W1538 residue and an intact local anesthetic site are required for lacosamide’s block of NaV1.7 at a clinically-achievable concentration. Moreover, the contribution of W1538 to lacosamide inhibitory effects appears to be isoform-specific.

Convergent Substitutions in a Sodium Channel Suggest Multiple Origins of Toxin Resistance in Poison Frogs.

Complex phenotypes typically have a correspondingly multifaceted genetic component. However, the genotype–phenotype association between chemical defense and resistance is often simple: genetic changes in the binding site of a toxin alter how it affects its target. Some toxic organisms, such as poison frogs (Anura: Dendrobatidae), have defensive alkaloids that disrupt the function of ion channels, proteins that are crucial for nerve and muscle activity. Using protein-docking models, we predict that three major classes of poison frog alkaloids (histrionicotoxins, pumiliotoxins, and batrachotoxins) bind to similar sites in the highly conserved inner pore of the muscle voltage-gated sodium channel, Nav1.4. We predict that poison frogs are somewhat resistant to these compounds because they have six types of amino acid replacements in the Nav1.4 inner pore that are absent in all other frogs except for a distantly related alkaloid-defended frog from Madagascar, Mantella aurantiaca. Protein-docking models and comparative phylogenetics support the role of these replacements in alkaloid resistance. Taking into account the four independent origins of chemical defense in Dendrobatidae, phylogenetic patterns of the amino acid replacements suggest that 1) alkaloid resistance in Nav1.4 evolved independently at least seven times in these frogs, 2) variation in resistance-conferring replacements is likely a result of differences in alkaloid exposure across species, and 3) functional constraint shapes the evolution of the Nav1.4 inner pore. Our study is the fi rst to demon strate the genetic basis of autoresistance in frogs with alkaloid defenses.

Progress in understanding slow inactivation speeds up

A new study reveals that conformational flexibility in the pore of a voltage-gated sodium channel may underlie slow inactivation.

Predicting Structural Details of the Sodium Channel Pore Basing on Animal Toxin Studies.

Eukaryotic voltage-gated sodium channels play key roles in physiology and are targets for many toxins and medically important drugs. Physiology, pharmacology, and general architecture of the channels has long been the subject of intensive research in academia and industry. In particular, animal toxins such as tetrodotoxin, saxitoxin, and conotoxins have been used as molecular probes of the channel structure. More recently, X-ray structures of potassium and prokaryotic sodium channels allowed elaborating models of the toxin-channel complexes that integrated data from biophysical, electrophysiological, and mutational studies. Atomic level cryo-EM structures of eukaryotic sodium channels, which became available in 2017, show that the selectivity filter structure and other important features of the pore domain have been correctly predicted. This validates further employments of toxins and other small molecules as sensitive probes of fine structural details of ion channels.

The voltage-gated sodium channel pore exhibits conformational flexibility during slow inactivation.

Slow inactivation in voltage-gated sodium channels (NaVs) directly regulates the excitability of neurons, cardiac myocytes, and skeletal muscles. Although NaV slow inactivation appears to be conserved across phylogenies-from bacteria to humans-the structural basis for this mechanism remains unclear. Here, using site-directed labeling and EPR spectroscopic measurements of membrane-reconstituted prokaryotic NaV homologues, we characterize the conformational dynamics of the selectivity filter region in the conductive and slow-inactivated states to determine the molecular events underlying NaV gating. Our findings reveal profound conformational flexibility of the pore in the slow-inactivated state. We find that the P1 and P2 pore helices undergo opposing movements with respect to the pore axis. These movements result in changes in volume of both the central and intersubunit cavities, which form pathways for lipophilic drugs that modulate slow inactivation. Our findings therefore provide novel insight into the molecular basis for state-dependent effects of lipophilic drugs on channel function.

Augmentation of pyrethrins content in callus of Chrysanthemum cinerariaefolium and establishing its insecticidal activity by molecular docking of NavMS Sodium Channel Pore receptor.

Pyrethrins are effective food-grade bio-pesticides obtained from the flowers of Chrysanthemum cinerariaefolium and this crop cannot be cultivated widely in India due to its specific agro-climatic requirement. Hence pyrethrins are mostly imported from Kenya. Therefore, the present study aims to develop a process for augmentation of pyrethrin contents in C. cinerariaefolium callus and establish the correlation between early knockdown effects through docking on grain storage insect. In vitro seedlings were used as explants to induce callus on MS medium with different concentrations of auxins and cytokinins. Pyrethrin extracted from the callus was estimated by RP-HPLC. In callus, total pyrethrin was found to be 17.5µg/g, which is higher than that found in natural flowers of certain Pyrethrum cultivars. The concentrations of cinerin II, pyrethrin II and jasmoline II were quite high in callus grown on solid medium. Bio-efficacy of pyrethrum extracts of flower and callus on insect Tribolium sp., showed higher repellency and early knock-down effect when compared with pure compound pestanal. Further, the rapid knockdown effect of all pyrethrins components was established by molecular docking studies targeting NavMS Sodium Channel Pore receptor docking followed bymultiple ligands simultaneous docking, performed to investigate the concurrent binding of different combinations of pyrethrin. Among the six pyrethrin components, the pyrethrin I and II were found to be a more efficient, binding more firmly to the target, exhibiting higher possibilities of insecticidal effect by an early knockdown mechanism.

Protonation state of inhibitors determines interaction sites within voltage-gated sodium channels

Voltage-gated sodium channels are essential for carrying electrical signals throughout the body, and mutations in these proteins are responsible for a variety of disorders, including epilepsy and pain syndromes. As such, they are the target of a number of drugs used for reducing pain or combatting arrhythmias and seizures. However, these drugs affect all sodium channel subtypes found in the body. Designing compounds to target select sodium channel subtypes will provide a new therapeutic pathway and would maximize treatment efficacy while minimizing side effects. Here, we examine the binding preferences of nine compounds known to be sodium channel pore blockers in molecular dynamics simulations. We use the approach of replica exchange solute tempering (REST) to gain a more complete understanding of the inhibitors' behavior inside the pore of NavMs, a bacterial sodium channel, and NavPas, a eukaryotic sodium channel. Using these simulations, we are able to show that both charged and neutral compounds partition into the bilayer, but neutral forms more readily cross it. We show that there are two possible binding sites for the compounds: (i) a site on helix 6, which has been previously determined by many experimental and computational studies, and (ii) an additional site, occupied by protonated compounds in which the positively charged part of the drug is attracted into the selectivity filter. Distinguishing distinct binding poses for neutral and charged compounds is essential for understanding the nature of pore block and will aid the design of subtype-selective sodium channel inhibitors.

Fenestration Mutants of a Voltage-Gated Sodium Channel that Modify Channel Blocker Ingress

The high-resolution crystal structure of the voltage-gated sodium channel pore of NavMs from M. marinus (Bagneris et al, 2014) was previously solved in the presence and absence of channel blocker drugs. In this study, mutants were designed, expressed and characterised in order to block the transmembrane fenestrations that are proposed to enable ingress of drugs into the hydrophobilic binding cavity, and which subsequently block sodium ion translocation through the pore. Guided by the HOLE analysis programme (Smart et al, 1996) we identified critical residues in helix S6 adjacent to the fenestration. We were then able to produce a number of mutants with side chains of different sizes, aimed at narrowing or blocking the fenestration, in order to prevent drug entry into the internal pore cavity. Using circular dichroism (CD) spectroscopy we identified whether the mutants had any differential effect on channel stability in the presence/absence of the drugs, and then we used X-ray crystallography to determine the high resolution structures of the fenestration mutants. Supported by grants from the BBSRC.

PH Modulation of Voltage-Gated Sodium Channels.

Changes in blood and tissue pH accompany physiological and pathophysiological conditions including exercise, cardiac ischemia, ischemic stroke, and cocaine ingestion. These conditions are known to trigger the symptoms of electrical diseases in patients carrying sodium channel mutations. Protons cause a diverse set of changes to sodium channel gating, which generally lead to decreases in the amplitude of the transient sodium current and increases in the fraction of non-inactivating channels that pass persistent currents. These effects are shared with disease-causing mutants in neuronal, skeletal muscle, and cardiac tissue and may be compounded in mutants that impart greater proton sensitivity to sodium channels, suggesting a role of protons in triggering acute symptoms of electrical disease.In this chapter, we review the mechanisms of proton block of the sodium channel pore and a suggested mode of action by which protons alter channel gating. We discuss the available data on isoform specificity of proton effects and tissue level effects. Finally, we review the role that protons play in disease and our own recent studies on proton-sensitizing mutants in cardiac and skeletal muscle sodium channels.

Novel derivatives of phthalimide with potent anticonvulsant activity in PTZ and MES seizure models.

Objective(s):Phthalimide-based derivatives have anticonvulsant activity like as phenytoin by inhibition of sodium channel. In our previously research we mentioned about some phthalimide derivatives as potent anticonvulsant agents.Materials and Methods:Fourteen analogs of 2-substituted phthalimide pharmacophore were synthesized and then were evaluated for the anticonvulsant activities in pentylenetetrazole-induced seizures (PTZ) and maximal electroshock seizure (MES) models.Results:The in vivo screening results showed that all the analogs have the ability to protect against the maximal electroshock and PTZ. The compounds 3 and 9 elevated clonic seizure thresholds at 30 min which were more active than the standard medicine phenytoin. Compounds 3, 6, 7, 11, 13 and 14 with 100% protection were the most potent ones in tonic seizure. The most potent compound in the both PTZ and MES models was compound 3. Using a model of the open pore of sodium channel, all of the compounds were docked. Results of docking showed that the ligands interacted mainly with residues II-S6 of NaV1.2 by making hydrogen bonds and have additional hydrophobic interactions with other domains in the channel’s inner pore.Conclusion:Some of these compounds are more potent than phenytoin simultaneously in the clonic and tonic seizures.

The Sodium Ion Binding Region at the Focus of P1 Helices Attracts both Charged and Electroneutral Ligands of Sodium Channels

The inner pore of eukaryotic voltage-gated sodium channels is targeted by ligands of dramatically different chemical structures. These include organic cations such as a local anesthetic lidocaine and electroneutral drugs such as an anticonvulsant carbamazepine. Mutations of the critical phenylalanine residue in helix IVS6 and some other inner pore-facing residues are known to affect action of both charged and neutral ligands. The structural cause of this ligand-binding promiscuity of sodium channels is unclear. Here we used the X-ray structure of a prokaryotic sodium channel NavMs to model the pore domain of the Nav1.x channels. We further employed the Monte Carlo energy-minimization method to perform intensive docking of lidocaine and carbamazepine from thousands starting points in the inner-pore region. The sodium ion NaIII, which is located between the four backbone carbonyls at the C-ends of P1 helices and does not make direct contacts with the channel protein, attracted carbamazepine, but repelled lidocaine. Therefore we further docked electroneutral ligands (lamotrigine, carbamazepine, phenytoin, lacosamide and bisphenol A) and cationic ligands (lidocaine, QX-314, cocaine, quinidine, and sipatrigine) in the channel models, respectively, with and without NaIII. In our models all the ligands interacted with the phenylalanine residue in IVS6 and most of the ligands also interacted with the tyrosine residue in IVS6. Some ligands extended their moieties in the III/IV sidewalk (fenestration). The electroneutral ligands bound the sodium ion with their electronegative groups and lacosamide chelated this ion. The ligand-bound ion remained close to the NaIII position due to attraction to the pore-facing backbone carbonyls. The same region attracted the charged group of the cationic ligands. In the predicted binding modes even small-size ligands would block the ion permeation by the electrostatic and steric mechanisms. Our study proposes a common pharmacophore for the diverse ligands. It includes a cation (the ligand's ammonium group or the ligand-bound sodium ion) and an aromatic moiety, which are usually linked by four bonds. Supported by NSERC and RFBR.

The Receptor Site and Mechanism of Action of Sodium Channel Blocker Insecticides

Sodium channels are excellent targets of both natural and synthetic insecticides with high insect selectivity. Indoxacarb, its active metabolite DCJW, and metaflumizone (MFZ) belong to a relatively new class of sodium channel blocker insecticides (SCBIs) with a mode of action distinct from all other sodium channel-targeting insecticides, including pyrethroids. Electroneutral SCBIs preferably bind to and trap sodium channels in the inactivated state, a mechanism similar to that of cationic local anesthetics. Previous studies identified several SCBI-sensing residues that face the inner pore of sodium channels. However, the receptor site of SCBIs, their atomic mechanisms, and the cause of selective toxicity of MFZ remain elusive. Here, we have built a homology model of the open-state cockroach sodium channel BgNav1-1a. Our computations predicted that SCBIs bind in the inner pore, interact with a sodium ion at the focus of P1 helices, and extend their aromatic moiety into the III/IV domain interface (fenestration). Using model-driven mutagenesis and electrophysiology, we identified five new SCBI-sensing residues, including insect-specific residues. Our study proposes the first three-dimensional models of channel-bound SCBIs, sheds light on the molecular basis of MFZ selective toxicity, and suggests that a sodium ion located in the inner pore contributes to the receptor site for electroneutral SCBIs.

Docking Simulation of the Binding Interactions of Saxitoxin Analogs Produced by the Marine Dinoflagellate Gymnodinium catenatum to the Voltage-Gated Sodium Channel Nav1.4.

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na+ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.

Point-Mutations in Skeletal Muscle Voltage-Gated Sodium Channels Confer Resistance to Tetrodotoxin: But at a Cost?

The interaction between toxic newts (Taricha) and several resistant garter snakes (Thamnophis) provides a model system for the study of predator-prey coevolution (Brodie & Brodie 1999). Pacific newts secrete through their skin the potent neurotoxin, tetrodotoxin (TTX, Fuhrman et al., 1964), a compound that specifically binds to the outer pore of voltage-gated sodium channels (NaV proteins, Fozzard et al., 1994). TTX-ligation prevents sodium ion movement across the membrane and therefore abolishes excitability in skeletal muscle and peripheral nerves (Hille, 2001). Three species of garter snake that prey on sympatric newts have adapted to these toxic newts by circumventing the block through several independently evolved mutations within the pore loop of the skeletal muscle channel variant, NaV1.4 (Feldman et al., 2012). These mutations reduce the affinity of TTX to the pore and provide physiological resistance (Feldman et al., 2009). However, these same mutations are also expected to alter the biophysical properties of the channel, in particular the permeation and selectivity (Hille, 2001). We show that animals with greater TTX resistance displayed diminished skeletal muscle performance (phasic and tetanic contractions). We are currently testing the hypothesis that changes in skeletal muscle sodium channel current density, activation, inactivation, and recovery could potentially explain the tissue-level findings.

Convergent Substitutions in a Sodium Channel Suggest Multiple Origins of Toxin Resistance in Poison Frogs.

Complex phenotypes typically have a correspondingly multifaceted genetic component. However, the genotype-phenotype association between chemical defense and resistance is often simple: genetic changes in the binding site of a toxin alter how it affects its target. Some toxic organisms, such as poison frogs (Anura: Dendrobatidae), have defensive alkaloids that disrupt the function of ion channels, proteins that are crucial for nerve and muscle activity. Using protein-docking models, we predict that three major classes of poison frog alkaloids (histrionicotoxins, pumiliotoxins, and batrachotoxins) bind to similar sites in the highly conserved inner pore of the muscle voltage-gated sodium channel, Nav1.4. We predict that poison frogs are somewhat resistant to these compounds because they have six types of amino acid replacements in the Nav1.4 inner pore that are absent in all other frogs except for a distantly related alkaloid-defended frog from Madagascar, Mantella aurantiaca. Protein-docking models and comparative phylogenetics support the role of these replacements in alkaloid resistance. Taking into account the four independent origins of chemical defense in Dendrobatidae, phylogenetic patterns of the amino acid replacements suggest that 1) alkaloid resistance in Nav1.4 evolved independently at least seven times in these frogs, 2) variation in resistance-conferring replacements is likely a result of differences in alkaloid exposure across species, and 3) functional constraint shapes the evolution of the Nav1.4 inner pore. Our study is the first to demonstrate the genetic basis of autoresistance in frogs with alkaloid defenses.

Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform.

This paper describes the use of a newly-developed micro-chip bilayer platform to examine the electrophysiological properties of the prokaryotic voltage-gated sodium channel pore (NavSp) from Silicibacter pomeroyi. The platform allows up to 6 bilayers to be analysed simultaneously. Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips. The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds. Dose-dependent modulation of the opening probability by the channel blocking drug nifedipine was measured and its IC50 determined.

Sodium Ion Coordination in the Selectivity Filter of a Voltage-Gated Sodium Channel

Voltage-gated sodium channels are essential for electrical signalling across eukaryotic cell membranes. They exhibit strong selectivity for sodium over other cations, thus enabling the finely-tuned cascade of events associated with action potentials. A new high resolution crystal structure of the prokaryotic sodium channel pore NavMs from Magnetococcus marinus provides the first view of the locations of sodium ions within the selectivity filter of a sodium channel. The structure reveals three sodium ions are bound within the selectivity filter. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and make no direct contact with the polypeptide backbone, instead there are interactions with conserved mainchain glutamate and serine residues, as well as backbone carbonyl atoms, all of which are likely mediated by a disordered hydration shell. Electrostatic calculations on the structure are compatible with relative cation selectivities of Na+≈Li+>>K+, Ca+, which correspond with the ion permeability ratios measured for these channels. Mutation of the conserved glutamate 178 to aspartate results in reduced sodium ion conductance through the pore. Interestingly, our structure of the E178D mutant reveals that it lacks the sodium ion nearest the extracellular vestibule and most closely associated with E178 in the native structure, explaining the reduced conductance. These results provide insight into the biophysical determinants of sodium-selectivity, which initiates the opening of other ion channels to shape the action potential waveform.

Efficient Characterization of Protein Cavities within Molecular Simulation Trajectories: trj_cavity.

Protein cavities and tunnels are critical in determining phenomena such as ligand binding, molecular transport, and enzyme catalysis. Molecular dynamics (MD) simulations enable the exploration of the flexibility and conformational plasticity of protein cavities, extending the information available from static experimental structures relevant to, for example, drug design. Here, we present a new tool (trj_cavity) implemented within the GROMACS ( www.gromacs.org ) framework for the rapid identification and characterization of cavities detected within MD trajectories. trj_cavity is optimized for usability and computational efficiency and is applicable to the time-dependent analysis of any cavity topology, and optional specialized descriptors can be used to characterize, for example, protein channels. Its novel grid-based algorithm performs an efficient neighbor search whose calculation time is linear with system size, and a comparison of performance with other widely used cavity analysis programs reveals an orders-of-magnitude improvement in the computational cost. To demonstrate its potential for revealing novel mechanistic insights, trj_cavity has been used to analyze long-time scale simulation trajectories for three diverse protein cavity systems. This has helped to reveal, respectively, the lipid binding mechanism in the deep hydrophobic cavity of a soluble mite-allergen protein, Der p 2; a means for shuttling carbohydrates between the surface-exposed substrate-binding and catalytic pockets of a multidomain, membrane-proximal pullulanase, PulA; and the structural basis for selectivity in the transmembrane pore of a voltage-gated sodium channel (NavMs), embedded within a lipid bilayer environment. trj_cavity is available for download under an open-source license ( http://sourceforge.net/projects/trjcavity ). A simplified, GROMACS-independent version may also be compiled.