Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

NaChBac is a bacterial voltage-gated sodium (Na_v) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Na_v channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of Na_vRh, a marine bacterial NaChBac ortholog. Two Na⁺ binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na⁺ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca²⁺ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na⁺ and Ca²⁺ to the vestibule of the SF. In addition, Na⁺ permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Na_v orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na⁺ over Ca²⁺ in mammalian Na_v channels.     

Structure of a Prokaryotic Sodium Channel Pore Reveals Essential Gating Elements and an Outer Ion Binding Site Common to Eukaryotic Channels

Voltage-gated sodium channels (Na_Vs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial Na_V (BacNa_V) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of Na_VAe1p, a pore-only BacNa_V derived from Na_VAe1, a BacNa_V from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNa_Vs show that two elements defined by the Na_VAe1p structure, an S6 activation gate position and the cytoplasmic tail “neck”, are central to BacNa_V gating. The structure also reveals the selectivity filter ion entry site, termed the “outer ion” site. Comparison with mammalian voltage-gated calcium channel (Ca_V) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of Ca_V high-affinity calcium binding. Our findings underscore commonalities between BacNa_Vs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.     

Binding modes of μ-conotoxin to the bacterial sodium channel (NaVAb)

Polypeptide toxins isolated from the venom of cone snails, known as μ-conotoxins, block voltage-gated sodium channels by physically occluding the ion-conducting pathway. Using molecular dynamics, we show that one subtype of μ-conotoxins, PIIIA, effectively blocks the bacterial voltage-gated sodium channel Na_VAb, whose crystal structure has recently been elucidated. The spherically shaped toxin, carrying a net charge of +6 e with six basic residues protruding from its surface, is attracted by the negatively charged residues on the vestibular wall and the selectivity filter of the channel. The side chain of each of these six arginine and lysine residues can wedge into the selectivity filter, whereas the side chains of other basic residues form electrostatic complexes with two acidic residues on the channel. We construct the profile of potential of mean force for the unbinding of PIIIA from the channel, and predict that PIIIA blocks the bacterial sodium channel with subnanomolar affinity.     

Protein arginine methyl transferases-3 and -5 increase cell surface expression of cardiac sodium channel

The α-subunit of the cardiac voltage-gated sodium channel (Na_V1.5) plays a central role in cardiomyocyte excitability. We have recently reported that Na_V1.5 is post-translationally modified by arginine methylation. Here, we aimed to identify the enzymes that methylate Na_V1.5, and to describe the role of arginine methylation on Na_V1.5 function. Our results show that protein arginine methyl transferase (PRMT)-3 and -5 methylate Na_V1.5 in vitro, interact with Na_V1.5 in human embryonic kidney (HEK) cells, and increase Na_V1.5 current density by enhancing Na_V1.5 cell surface expression. Our observations are the first evidence of regulation of a voltage-gated ion channel, including calcium, potassium, sodium and TRP channels, by arginine methylation.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Unexpected synergism between Tetrodotoxin and μ-conotoxin in blocking voltage-gated sodium channels

Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (Na_Vs). Like TTX, μ-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of neurotoxin receptor Site 1 of Na_Vs. Here, we report unexpected results showing that the recently discovered μ-conotoxin KIIIA and TTX can simultaneously bind to Site 1 and act in concert. Results with saturating concentrations of peptide applied to voltage-clamped Xenopus oocytes expressing brain Na_V1.2, and single-channel recordings from brain channels in lipid bilayers, show that KIIIA or its analog, KIIIA[K7A], block partially, with a residual current that can be completely blocked by TTX. In addition, the kinetics of block by TTX and peptide are each affected by the prior presence of the other toxin. For example, bound peptide slows subsequent binding of TTX (an antagonistic interaction) and slows TTX dissociation when both toxins are bound (a synergistic effect on block). The overall functional consequence resulting from the combined action of the toxins depends on the quantitative balance between these opposing actions. The results lead us to postulate that in the bi-liganded Na_V complex, TTX is bound between the peptide and the selectivity filter. These observations refine our view of Site 1 and open new possibilities in Na_V pharmacology.     

Enhancement of Sodium Current in NG108-15 Cells during Neural Differentiation is Mainly due to an Increase in NaV1.7 Expression

It is well known that morphological and functional changes during neural differentiation sometimes accompany the expression of various voltage-gated ion channels. In this work, we investigated whether the enhancement of sodium current in differentiated neuroblastoma × glioma NG108-15 cells treated with dibutyryl cAMP is related to the expression of voltage-gated sodium channels. The results were as follows. (1) Sodium current density on peak voltage in differentiated cells was significantly enhanced compared with that in undifferentiated cells, as detected by the whole-cell patch clamp method. The steady-state inactivation curve in differentiated cells was similar to that for undifferentiated cells, but a hyperpolarized shift in the activation curve for differentiated cells was observed. The sodium currents of differentiated and undifferentiated cells were completely inhibited by 10⁻⁷ M tetrodotoxin (TTX). (2) The only Na_V mRNA with an increased expression level during neuronal differentiation was that for Na_V1.7, as observed by real-time PCR analysis. (3) The increase in the level of Na_V1.7 α subunit expression during neuronal differentiation was also observed by immunocytochemistry; in particular, the localization of Na_V1.7 α subunits on the soma, varicosities and growth cone was significant. These results suggest that the enhancement of TTX-sensitive sodium current density in differentiated NG108-15 cells is mainly due to the increase in the expression of the TTX-sensitive voltage-gated Na⁺ channel, Na_V1.7.     

Molecular Dynamics Study of Binding of μ-Conotoxin GIIIA to the Voltage-Gated Sodium Channel Nav1.4

Homology models of mammalian voltage-gated sodium (Na_V) channels based on the crystal structures of the bacterial counterparts are needed to interpret the functional data on sodium channels and understand how they operate. Such models would also be invaluable in structure-based design of therapeutics for diseases involving sodium channels such as chronic pain and heart diseases. Here we construct a homology model for the pore domain of the Na_V1.4 channel and use the functional data for the binding of µ-conotoxin GIIIA to Na_V1.4 to validate the model. The initial poses for the Na_V1.4–GIIIA complex are obtained using the HADDOCK protein docking program, which are then refined in molecular dynamics simulations. The binding mode for the final complex is shown to be in broad agreement with the available mutagenesis data. The standard binding free energy, determined from the potential of mean force calculations, is also in good agreement with the experimental value. Because the pore domains of Na_V1 channels are highly homologous, the model constructed for Na_V1.4 will provide an excellent template for other Na_V1 channels.     

Structural and functional insights into the inhibition of human voltage-gated sodium channels by μ-conotoxin KIIIA disulfide isomers

μ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (Na_V) subtypes, including Na_V1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a ‘native’ CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at Na_V1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and Na_V subtype selectivity across human Na_V channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits Na_V subtypes with a rank order of potency of Na_V1.4 > 1.2 > 1.7 and Isomer 2 in the order of Na_V1.4≈1.2 > 1.7, while the native isomer inhibited Na_V1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNa_V1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant Na_V subtypes.     

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaVAb channel

Ca_V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca²⁺ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca_VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca²⁺ ions in the Ca_VAb channels in presence and absence of an external field. Exposing Ca_VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca²⁺ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca²⁺ ions binding in the selectivity filter may interrupt the mechanism of Ca²⁺ ion conduction, and leads to a reduction of Ca²⁺ ion permeation through the Ca_VAb channel.     

Models of the structure and gating mechanisms of the pore domain of the NaChBac ion channel

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K_V and Ca_V channels. Like K_V channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca_V channels. We developed structural models of the NaChBac channel in closed and open conformations, using K⁺-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K⁺ crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K⁺ channels of known structure and Na_V, Ca_V, and TRP channels of unknown structure.     

Convergent adaptation to dangerous prey proceeds through the same first‐step mutation in the garter snake Thamnophis sirtalis

Convergent phenotypes often result from similar underlying genetics, but recent work suggests convergence may also occur in the historical order of substitutions en route to an adaptive outcome. We characterized convergence in the mutational steps to two independent outcomes of tetrodotoxin (TTX) resistance in separate geographic lineages of the common garter snake (Thamnophis sirtalis) that coevolved with toxic newts. Resistance is largely conferred by amino acid changes in the skeletal muscle sodium channel (Na_V1.4) that interfere with TTX‐binding. We sampled variation in Na_V1.4 throughout western North America and found clear evidence that TTX‐resistant changes in both lineages began with the same isoleucine‐valine mutation (I1561V) within the outer pore of Na_V1.4. Other point mutations in the pore, shown to confer much greater resistance, accumulate later in the evolutionary progression and always occur together with the initial I1561V change. A gene tree of Na_V1.4 suggests the I1561V mutations in each lineage are not identical‐by‐decent, but rather they arose independently. Convergence in the evolution of channel resistance is likely the result of shared biases in the two lineages of T. sirtalis—only a few mutational routes can confer TTX resistance while maintaining the conserved function of voltage‐gated sodium channels.     

Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na(v)). The skeletal muscle Na(v) (Na(v)1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na(v)1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na(v)1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na(v)1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Folding similarity of the outer pore region in prokaryotic and eukaryotic sodium channels revealed by docking of conotoxins GIIIA,PIIIA, and KIIIA in a NavAb-based model of Nav1.4

Voltage-gated sodium channels are targets for many drugs and toxins. However, the rational design of medically relevant channel modulators is hampered by the lack of x-ray structures of eukaryotic channels. Here, we used a homology model based on the x-ray structure of the NavAb prokaryotic sodium channel together with published experimental data to analyze interactions of the μ-conotoxins GIIIA, PIIIA, and KIIIA with the Nav1.4 eukaryotic channel. Using Monte Carlo energy minimizations and published experimentally defined pairwise contacts as distance constraints, we developed a model in which specific contacts between GIIIA and Nav1.4 were readily reproduced without deformation of the channel or toxin backbones. Computed energies of specific interactions between individual residues of GIIIA and the channel correlated with experimental estimates. The predicted complexes of PIIIA and KIIIA with Nav1.4 are consistent with a large body of experimental data. In particular, a model of Nav1.4 interactions with KIIIA and tetrodotoxin (TTX) indicated that TTX can pass between Nav1.4 and channel-bound KIIIA to reach its binding site at the selectivity filter. Our models also allowed us to explain experimental data that currently lack structural interpretations. For instance, consistent with the incomplete block observed with KIIIA and some GIIIA and PIIIA mutants, our computations predict an uninterrupted pathway for sodium ions between the extracellular space and the selectivity filter if at least one of the four outer carboxylates is not bound to the toxin. We found a good correlation between computational and experimental data on complete and incomplete channel block by native and mutant toxins. Thus, our study suggests similar folding of the outer pore region in eukaryotic and prokaryotic sodium channels.     

Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics

Despite the availability of several crystal structures of bacterial voltage-gated Na⁺ channels, the structure of eukaryotic Na⁺ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNa_V1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na⁺ channels predict that the side chain of rNa_V1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na⁺ channels.     

A novel homology model of TRPC3 reveals allosteric coupling between gate and selectivity filter

Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (Na_VAB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629–631 as the narrowest part of the permeation pathway with an estimated pore diameter of <5.8 Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in Na_VAB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca²⁺ permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channel's selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3.     

Ancient Origin of Four-Domain Voltage-gated Na<Superscript>+</Superscript> Channels Predates the Divergence of Animals and Fungi

The four-domain voltage-gated Na⁺ channels are believed to have arisen in multicellular animals, possibly during the evolution of the nervous system. Recent genomic studies reveal that many ion channels, including Na⁺ channels and Ca²⁺ channels previously thought to be restricted to animals, can be traced back to one of the unicellular ancestors of animals, Monosiga brevicollis. The eukaryotic supergroup Opisthokonta contains animals, fungi, and a diverse group of their unicellular relatives including M. brevicollis. Here, we demonstrate the presence of a putative voltage-gated Na⁺ channel homolog (TtrNa_V) in the apusozoan protist Thecamonas trahens, which belongs to the unicellular sister group to Opisthokonta. TtrNa_V displays a unique selectivity motif distinct from most animal voltage-gated Na⁺ channels. The identification of TtrNa_V suggests that voltage-gated Na⁺ channels might have evolved before the divergence of animals and fungi. Furthermore, our analyses reveal that Na_V channels have been lost independently in the amoeboid holozoan Capsaspora owczarzaki of the animal lineage and in several basal fungi. These findings provide novel insights into the evolution of four-domain voltage-gated ion channels, ion selectivity, and membrane excitability in the Opisthokonta lineage.     

Structural Pharmacology of Voltage-Gated Sodium Channels

Voltage-gated sodium (Na_V) channels initiate and propagate action potentials in excitable tissues to mediate key physiological processes including heart contraction and nervous system function. Accordingly, Na_V channels are major targets for drugs, toxins and disease-causing mutations. Recent breakthroughs in cryo-electron microscopy have led to the visualization of human Na_V1.1, Na_V1.2, Na_V1.4, Na_V1.5 and Na_V1.7 channel subtypes at high-resolution. These landmark studies have greatly advanced our structural understanding of channel architecture, ion selectivity, voltage-sensing, electromechanical coupling, fast inactivation, and the molecular basis underlying Na_V channelopathies. Na_V channel structures have also been increasingly determined in complex with toxin and small molecule modulators that target either the pore module or voltage sensor domains. These structural studies have provided new insights into the mechanisms of pharmacological action and opportunities for subtype-selective Na_V channel drug design. This review will highlight the structural pharmacology of human Na_V channels as well as the potential use of engineered and chimeric channels in future drug discovery efforts.