Bacterial Nav Channels Research Articles

Molecular Dynamics Simulations of Ion Permeation in Human Voltage-Gated Sodium Channels.

The recent determination of cryo-EM structures of voltage-gated sodium (Nav) channels has revealed many details of these proteins. However, knowledge of ionic permeation through the Nav pore remains limited. In this work, we performed atomistic molecular dynamics (MD) simulations to study the structural features of various neuronal Nav channels based on homology modeling of the cryo-EM structure of the human Nav1.4 channel and, in addition, on the recently resolved configuration for Nav1.2. In particular, single Na+ permeation events during standard MD runs suggest that the ion resides in the inner part of the Nav selectivity filter (SF). On-the-fly free energy parametrization (OTFP) temperature-accelerated molecular dynamics (TAMD) was also used to calculate two-dimensional free energy surfaces (FESs) related to single/double Na+ translocation through the SF of the homology-based Nav1.2 model and the cryo-EM Nav1.2 structure, with different realizations of the DEKA filter domain. These additional simulations revealed distinct mechanisms for single and double Na+ permeation through the wild-type SF, which has a charged lysine in the DEKA ring. Moreover, the configurations of the ions in the SF corresponding to the metastable states of the FESs are specific for each SF motif. Overall, the description of these mechanisms gives us new insights into ion conduction in human Nav cryo-EM-based and cryo-EM configurations that could advance understanding of these systems and how they differ from potassium and bacterial Nav channels.

Determinants of conductance of a bacterial voltage-gated sodium channel

Through molecular dynamics (MD) and free energy simulations in electric fields, we examine the factors influencing conductance of bacterial voltage-gated sodium channel NavMs. The channel utilizes four glutamic acid residues in the selectivity filter (SF). Previously, we have shown, through constant pH and free energy calculations of pKa values, that fully deprotonated, singly protonated, and doubly protonated states are all feasible at physiological pH, depending on how many ions are bound in the SF. With 173 MD simulations of 450 or 500 ns and additional free energy simulations, we determine that the conductance is highest for the deprotonated state and decreases with each additional proton bound. We also determine that the pKa value of the four glutamic residues for the transition between deprotonated and singly protonated states is close to the physiological pH and that there is a small voltage dependence. The pKa value and conductance trends are in agreement with experimental work on bacterial Nav channels, which show a decrease in maximal conductance with lowering of pH, with pKa in the physiological range. We examine binding sites for Na+ in the SF, compare with previous work, and note a dependence on starting structures. We find that narrowing of the gate backbone to values lower than the crystal structure's backbone radius reduces the conductance, whereas increasing the gate radius further does not affect the conductance. Simulations with some amount of negatively charged lipids as opposed to purely neutral lipids increases the conductance, as do simulations at higher voltages.

Conservation and divergence in NaChBac and NaV1.7 pharmacology reveals novel drug interaction mechanisms

Voltage-gated Na+ (NaV) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts, bacterial NaV channels possess a simpler, fourfold symmetric structure and have facilitated studies of the structural basis of channel gating. However, the pharmacology of bacterial NaV remains largely unexplored. Here we systematically screened 39 NaV modulators on a bacterial channel (NaChBac) and characterized a selection of compounds on NaChBac and a mammalian channel (human NaV1.7). We found that while many compounds interact with both channels, they exhibit distinct functional effects. For example, the local anesthetics ambroxol and lidocaine block both NaV1.7 and NaChBac but affect activation and inactivation of the two channels to different extents. The voltage-sensing domain targeting toxin BDS-I increases NaV1.7 but decreases NaChBac peak currents. The pore binding toxins aconitine and veratridine block peak currents of NaV1.7 and shift activation (aconitine) and inactivation (veratridine) respectively. In NaChBac, they block the peak current by binding to the pore residue F224. Nonetheless, aconitine has no effect on activation or inactivation, while veratridine only modulates activation of NaChBac. The conservation and divergence in the pharmacology of bacterial and mammalian NaV channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.

Covalent linkage of bacterial voltage-gated sodium channels

BackgroundBacterial sodium channels are important models for understanding ion permeation and selectivity. However, their homotetrameric structure limits their use as models for understanding the more complex eukaryotic voltage-gated sodium channels (which have a pseudo-heterotetrameric structure formed from an oligomer composed of four domains). To bridge this gap we attempted to synthesise oligomers made from four covalently linked bacterial sodium channel monomers and thus resembling their eukaryotic counterparts.ResultsWestern blot analyses revealed NaChBac oligomers to be inherently unstable whereas intact expression of NavMs oligomers was possible. Immunodectection using confocal microscopy and electrophysiological characterisation of NavMs tetramers confirmed plasma membrane localisation and equivalent functionality with wild type NavMs channels when expressed in human embryonic kidney cells.ConclusionThis study has generated new tools for the investigation of eukaryotic channels. The successful covalent linkage of four bacterial Nav channel monomers should permit the introduction of radial asymmetry into the structure of bacterial Nav channels and enable the known structures of these channels to be used to gain unique insights into structure-function relationships of their eukaryotic counterparts.

Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels.

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

An Open State Model of the Navab Channel Explored by Rosetta and Molecular Dynamics Simulation

Voltage-gated sodium (Nav) channels play a pivotal role in propagating electrical signals in excitable cells and key targets for development of novel therapeutics. Despite recent progress in determining x-ray structures of bacterial Nav channels in closed, inactivated, and partially open states, we are still missing a stable open state structure of a Nav channel that will be useful to study ion conduction, channel gating and drug - channel interactions. We used Rosetta molecular modeling software to build the full-length open state model of the bacterial NavAb channel. The fully activated state of the NavAb voltage sensor domain (VSD) was modeled using the crystal structure of NavRh channel VSD as a template. The initial open state of NavAb pore was modeled using the crystal structure of NavMs with partially open pore. Rebuilding the interacting regions of NavAb (S3, S4, S5, S6, and S4-S5 linker) using Rosetta loop modeling and relax approaches was essential to generate stable NavAb open state model. Output models were evaluated using RosettaMembrane energy function and Rosetta clustering approach. The top Rosetta models represented different alternative open states of the NavAb channel. We have conducted a set of molecular dynamics (MD) simulations on the NavAb channel open state model using the Anton supercomputer to examine its stability. The simulations revealed that the NavAb channel open state model reproducibly sustained a fully hydrated open pore that conducted sodium ions under an applied membrane potential for periods of hundreds of nanoseconds. These results suggest that our structural modeling approach could be useful for modeling multiple states of other ion channels and for rational design of novel therapeutics targeting ion channels.

Comparison of Ion Selectivity Mechanisms in Bacterial and Mammalian Sodium Channels

Voltage-gated sodium (Nav) channels play essential roles in electrical signalling in the body and are associated with many physiological disorders. The availability of recent high-resolution structures for bacterial Nav channels makes them ideal tools for exploring functional mechanisms. Bacterial and mammalian channels have been proposed to select for Na+ over K+ via different mechanisms, owing to their distinct selectivity signature sequences, with bacterial channels making use of a 4-fold ring of Glu side chains, in place of Asp, Glu, Lys and Ala (DEKA) in mammalian channels.

Molecular Modeling of Mammalian Nav1.4 Channel

Cell membranes consist of two layers of lipid molecules and are impermeable to ions. Ion transportation across the membrane is mediated via membrane proteins which are pumps, transporters and ion channels. Voltage gated sodium (NaV) channels are essential elements in ion channels family that responsible for the rapid upstroke of the action potential. The crystal structures of bacterial NaV channels that have been determined recently, have made way for studies of mammalian NaV channels through homology modeling.

Computational Study of Binding of μ-Conotoxin GIIIA to Bacterial Sodium Channels NaVAb and NaVRh.

Structures of several voltage-gated sodium (NaV) channels from bacteria have been determined recently, but the same feat might not be achieved for the mammalian counterparts in the near future. Thus, at present, computational studies of the mammalian NaV channels have to be performed using homology models based on the bacterial crystal structures. A successful homology model for the mammalian NaV1.4 channel was recently constructed using the extensive mutation data for binding of μ-conotoxin GIIIA to NaV1.4, which was further validated through studies of binding of other μ-conotoxins and ion permeation. Understanding the similarities and differences between the bacterial and mammalian NaV channels is an important issue, and the NaV1.4-GIIIA system provides a good opportunity for such a comparison. To this end, we study the binding of GIIIA to the bacterial channels NaVAb and NaVRh. The complex structures are obtained using docking and molecular dynamics simulations, and the dissociation of GIIIA is studied through umbrella sampling simulations. The results are compared to those obtained from the NaV1.4-GIIIA system, and the differences in the binding modes arising from the changes in the selectivity filters are highlighted.

Binary architecture of the Nav1.2-β2 signaling complex.

To investigate the mechanisms by which β-subunits influence Nav channel function, we solved the crystal structure of the β2 extracellular domain at 1.35Å. We combined these data with known bacterial Nav channel structural insights and novel functional studies to determine the interactions of specific residues in β2 with Nav1.2. We identified a flexible loop formed by (72)Cys and (75)Cys, a unique feature among the four β-subunit isoforms. Moreover, we found that (55)Cys helps to determine the influence of β2 on Nav1.2 toxin susceptibility. Further mutagenesis combined with the use of spider toxins reveals that (55)Cys forms a disulfide bond with (910)Cys in the Nav1.2 domain II pore loop, thereby suggesting a 1:1 stoichiometry. Our results also provide clues as to which disulfide bonds are formed between adjacent Nav1.2 (912/918)Cys residues. The concepts emerging from this work will help to form a model reflecting the β-subunit location in a Nav channel complex.

Computational Study of Binding of μ-Conotoxin GIIIA (μ-CTX) to Navrh and NavAb

Voltage gated sodium channels (Nav) are involved in several physiological activities of different cells from bacteria to mammalian. Since 2011, different crystal structures of bacterial-Nav channels have been published. Studying the interaction of toxins with bacterial-Nav channels could provide some understanding about using bacterial-Nav channels as a model of mammalian-Nav channels. Here, we studied the interaction of μ-Conotoxin-GIIIA (μ-GIIIA) with NavRh as well as with NavAb and compared the results with μ-GIIIA binding to Nav1.4.The crystal structures of NavRh (pdb id-4DXW) and NavAb (pdb id-4EKW) were embedded in a lipid bilayer and simulated for 50 ns separately.First blind docking of μ-GIIIA to pore domain of channels was done seperately using HADDDOCK to find the possible binding mode(s) and strongly interacting pairs were used in restraint docking.The best complex was selected according to the energy score. The binding pose of the toxin was transferred to a channel simulated in the membrane and the system was run for another 50 ns for stability. In NavRh, Arg-13 inserted to the pore and interacted with the S-ring while Lys11,Lys16 interacted with the E-ring while in NavAb, Lys-11 inserted to the pore and interacted with the E-ring while Arg-13, Lys-16 interacted with the surface of pore domain. The affinity of the complexes were calculated using the potential of mean force (PMF) and compared to the results for binding of μ-GIIIA to Nav1.4 which is previously done in our group.Binding modes of μ-GIIIA to bacterial channels are not similar to binding of μ-GIIIA to Nav1.4 although interacting residues of toxin are the same. PMF well depths indicate less affinity of μ-GIIIA to bacterial channels compared to Nav1.4. We conclude that bacterial Nav channels are not suitable to study human Nav channels.

Structural Basis of Nav1.7 Inhibition by an Isoform-Selective Small Molecule Antagonist

Voltage-gated sodium (Nav) channels are therapeutic targets for many cardiovascular and neurological disorders. Unfortunately, selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Recently, a series of highly selective small-molecule Nav channel antagonists (the aryl-sulfonamide series) has been identified that is able to achieve isoform selectivity by binding to the relatively poorly-conserved surface of voltage-sensor domain IV (VSD4). Understanding how these antagonists work and the details of their binding site has been a recent focus of the pharmaceutical industry which has hopes of identifying selective inhibitors of Nav1.7, an important target for novel pain drugs. Here, we describe the structural basis of Nav1.7 inhibition by this novel class of small molecule antagonist using X-ray crystallography, and thus present important experimental structures of a gating modifier in complex with a voltage-gated ion channel. To enable these unique mammalian Nav channel crystal structures, we exploited the established portability of VSDs and the presumed structural relatedness between human and bacterial Nav channels to develop a robust protein production and crystallization strategy. GX-936 and related aryl-sulfonamide inhibitors bind to the activated state of VSD4, where their anionic aryl sulfonamide warhead directly engages the R4 gating charge on the S4 helix. By opposing VSD4 deactivation, which stabilizes inactivated states of the channel, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism. Residues from the S2 and S3 helices are key determinants of isoform selectivity and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results shed light on the molecular basis of voltage sensing and establish the first structural blueprints to design selective Nav channel antagonists.

K+ Block Is the Mechanism of Functional Asymmetry in Bacterial Na(v) Channels.

Crystal structures of several bacterial Nav channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Nav channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial NavAb channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Nav channels. The non-equilibrium simulations indicate that two or three extracellular K+ ions can block the entrance to the selectivity filter of NavAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K+ ions that can be ‘locked’ in place by modest applied forces. In contrast to K+, three Na+ ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na+ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K+ block is equivalent to large applied potentials experimentally measured for two bacterial Nav channels to induce inward currents of K+ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.

Simulation Studies of Ion Permeation and Selectivity in Voltage-Gated Sodium Channels.

Voltage-gated ion channels are responsible for the generation and propagation of action potentials in electrically excitable cells. Molecular dynamics simulations have become a useful tool to study the molecular basis of ion transport in atomistic models of voltage-gated ion channels. The elucidation of several three-dimensional structures of bacterial voltage-gated sodium channels (Nav) in 2011 and 2012 opened the way to detailed computational investigations of this important class of membrane proteins. Here we review the numerous simulation studies of Na(+) permeation and selectivity in bacterial Nav channels published in the past 5years. These studies use a variety of simulation methodologies differing in force field parameters, molecular models, sampling algorithms, and simulation times. Although results disagree on the details of ion permeation mechanisms, they concur in the presence of two primary Na(+) binding sites in the selectivity filter and support a loosely coupled knock-on mechanism of Na(+) permeation. Comparative studies of Na(+), K(+), and Ca(2+) permeation reveal sites within Nav channels that are Na(+) selective, yet a consensus model of selectivity has not been established. We discuss the agreement between simulation and experimental resultsand propose strategies that may be used to resolve discrepancies between simulation studies in order to improve future computational studies of permeation and selectivity in ion channels.

Mechanism of Ion Permeation in Mammalian Voltage-Gated Sodium Channels.

Recent determination of the crystal structures of bacterial voltage-gated sodium (NaV) channels have raised hopes that modeling of the mammalian counterparts could soon be achieved. However, there are substantial differences between the pore domains of the bacterial and mammalian NaV channels, which necessitates careful validation of mammalian homology models constructed from the bacterial NaV structures. Such a validated homology model for the NaV1.4 channel was constructed recently using the extensive mutagenesis data available for binding of μ-conotoxins. Here we use this NaV1.4 model to study the ion permeation mechanism in mammalian NaV channels. Linking of the DEKA residues in the selectivity filter with residues in the neighboring domains is found to be important for keeping the permeation pathway open. Molecular dynamics simulations and potential of mean force calculations reveal that there is a binding site for a Na+ ion just inside the DEKA locus, and 1–2 Na+ ions can occupy the vestibule near the EEDD ring. These sites are separated by a low free energy barrier, suggesting that inward conduction occurs when a Na+ ion in the vestibule goes over the free energy barrier and pushes the Na+ ion in the filter to the intracellular cavity, consistent with the classical knock-on mechanism. The NaV1.4 model also provides a good description of the observed Na+/K+ selectivity.

Molecular Dynamics Simulations Describe the Mechanism of K Block in Bacterial Nav Channels

Although extensive electrophysiological characterization is available for eukaryotic voltage-gated Na+ channels (Nav), no high-resolution structures of these channels are available. Crystal structures of several bacterial Nav channels have been published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of the eukaryotic channels. Unlike eukaryotic Nav channels, however, the bacterial Nav channels are strongly outwardly rectifying, and the mechanism of this rectification has not previously been described. We used step-wise pulling protocols to implement Jarzynski's Equality in non-equilibrium molecular dynamics simulations of ion permeation through the bacterial NavAb channel to obtain a mechanistic description of this outward rectification. Results of the simulations indicate that two or three extracellular K+ ions bind tightly at the same z-coordinate along the selectivity filter of NavAb and can effectively block the channel in the presence of modest voltages or concentration driving forces. The configuration with two potassium ions located at the same z-coordinate is also found in the two-dimensional potential of mean forces generated from umbrella sampling and weighted histogram analysis. In contrast to K+, three Na+ ions move through the selectivity filter together as a unit in a “knock-on” mechanism of permeation. Differences in the amount of work required to move three Na+ ions through the selectivity filter of NavAb compared to three K+ ions predict the large negative reversal potentials observed for bacterial Nav channels in instantaneous current-voltage plots. The results of the simulations suggest that the block of bacterial voltage-gated Na+ channels by extracellular K+ does not occur in eukaryotic voltage-gated Na+ channels because of differences in the amino acids present in the selectivity filters of the different channels.

“Use Dependence” Without a Ball and Chain - Inhibition of Bacterial Sodium Channels by μ-Contoxins

“Use-” or “state-” dependent block of voltage-gated sodium channels is characteristic of therapeutic agents used to moderate electrical activity in pathologically hyperactive tissues and combat certain forms of epilepsy, cardiac arrhythmia, myotonia, and pain. Externally acting Na channel pore blockers are not commonly used as therapeutic drugs, but intraperitoneal μ-conotoxin (μCTX) KIIIA can act as an effective analgesic against inflammatory pain in mice, at concentrations that do not cause obvious systemic side effects (Zhang et al 2007 J Biol Chem 42:30699). This action appears to result from highly specific targeting to neuronal Na channels, without essential reliance on the use-dependent potency that contributes to the efficacy of clinical local anesthetics. μCTXs, although highly selective among closely related eukaryotic Na channels, have recently been found to block bacterial Nav channels with high affinity (Chen & Chung 2012 Biophys J 102:483; Finol-Urdaneta et al 2013 Biophys J 104:136a). In addition, the block was potentiated at more depolarized holding potentials. Here, we explore the dependence of μCTX PIIIA block of NaChBac and NavSp1 on varying patterns of conditioning depolarization. Briefly, in the low picomolar range, PIIIA application was associated with faster inactivation decay during a test depolarization, and block of the peak current. The speeding of inactivation precedes the block of current following PIIIA application, and this kinetic effect was obvious at more negative holding potentials than was the decrease in peak current. In NaChBac, both activation and inactivation were shifted in the hyperpolarizing direction by ∼25-30 mV in the presence of 5pM PIIIA. The data suggest that PIIIA binding is conformation dependent, with high affinity for non-conducting, depolarization-induced states. At present, the relative importance of “pre-activated” and “in-activated” states in potentiating inhibition by PIIIA is unclear.

Structural Modeling of Local Anesthetic Binding to the Pore-Domain of Human Nav1.5 in Open and Closed States using Rosetta

Mutations in voltage-gated sodium (Nav) channel isoforms are correlated with a wide range of cardiovascular and neurological diseases in humans, and are therefore are important targets for the rational design of novel drugs. The cardiac isoform of the Nav channel, Nav1.5, presents a unique target for the development of antiarrhythmic drugs. In this work, we identify key structural motifs for local anesthetic binding in the pore-domain of the open and closed states of the human Nav1.5 channel. The Rosetta structural modeling method was used to construct models of human Nav1.5 isoform based on the 3D crystal structures of bacterial Nav channels: NavRh (closed state) and NavMs (open state). The resulting lowest free-energy models were selected for local anesthetic docking simulations. Rosetta loop modeling and global relaxation of 10,000 models yielded a convergent motif in the selectivity filter region of a stabilizing hydrogen bond network between Tryptophan and Threonine pairs. A membrane-facing fenestration near the S6 helix of domain IV and the S5 helix of domain III was also structurally conserved, and is a proposed site of neutral drug entry. Docking simulations of the channel blocker, lidocaine, reveal key protein-ligand binding configurations within the pore. Our preliminary models of the human Nav1.5 channel in the open and closed states reveal highly conserved structural motifs important for both stabilization of the pore domain, as well as for drug entry and binding. Future work will use structural models of drug interaction with human Nav1.5 as a dynamic testing platform for the calculation of the kinetics of drug binding and unbinding.

Ion selectivity strategies of sodium channel selectivity filters.

CONSPECTUS: Sodium ion channels selectively transport Na(+) cations across the cell membrane. These integral parts of the cell machinery are implicated in regulating the cardiac, skeletal and smooth muscle contraction, nerve impulses, salt and water homeostasis, as well as pain and taste perception. Their malfunction often results in various channelopathies of the heart, brain, skeletal muscles, and lung; thus, sodium channels are key drug targets for various disorders including cardiac arrhythmias, heart attack, stroke, migraine, epilepsy, pain, cancer, and autoimmune disorders. The ability of sodium channels to discriminate the native Na(+) among other competing ions in the surrounding fluids is crucial for proper cellular functions. The selectivity filter (SF), the narrowest part of the channel's open pore, lined with amino acid residues that specifically interact with the permeating ion, plays a major role in determining Na(+) selectivity. Different sodium channels have different SFs, which vary in the symmetry, number, charge, arrangement, and chemical type of the metal-ligating groups and pore size: epithelial/degenerin/acid-sensing ion channels have generally trimeric SFs lined with three conserved neutral serines and/or backbone carbonyls; eukaryotic sodium channels have EKEE, EEKE, DKEA, and DEKA SFs with an invariant positively charged lysine from the second or third domain; and bacterial voltage-gated sodium (Nav) channels exhibit symmetrical EEEE SFs, reminiscent of eukaryotic voltage-gated calcium channels. How do these different sodium channel SFs achieve high selectivity for Na(+) over its key rivals, K(+) and Ca(2+)? What factors govern the metal competition in these SFs and which of these factors are exploited to achieve Na(+) selectivity in the different sodium channel SFs? The free energies for replacing K(+) or Ca(2+) bound inside different model SFs with Na(+), evaluated by a combination of density functional theory and continuum dielectric calculations, have shed light on these questions. The SFs of epithelial and eukaryotic Nav channels select Na(+) by providing an optimal number and ligating strength of metal ligands as well as a rigid pore whose size fits the cognate Na(+) ideally. On the other hand, the SFs of bacterial Nav channels select Na(+), as the protein matrix attenuates ion-protein interactions relative to ion-solvent interactions by enlarging the pore and allowing water to enter, so the ion interacts indirectly with the conserved glutamates via bridging water molecules. This shows how these various SFs have adapted to the specific physicochemical properties of the native ion, using different strategies to select Na(+) among its contenders.

Modulation of a voltage-gated Na+ channel by sevoflurane involves multiple sites and distinct mechanisms.

Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na(+) (NaV) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial NaV channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.