Sodium Channel Pore Research Articles

Modeling the Outer Pore of Sodium and Calcium Channels

In the absence of x-ray structures of sodium and calcium channels, their homology models based on x-ray structures of potassium channels are used to design and rationalize experiments. A challenge is to model the outer-pore region that folds differently from potassium channels. Here we report a model of the outer-pore region of NaV1.4, which is based on a large body of experimental data, including specific contacts of toxins with individual channel residues. The model inherits from our previous model the general disposition of the P-helices, the selectivity-filter residues, and the outer carboxylates, but provides a novel view on the role of other highly conserved residues in the outer pore. In the absence of secondary-structure elements, structural stability of the outer pore should be supported by specific contacts. We propose a network of such contacts including intra- and inter-domain H-bonds, knob-into-the-hole contacts, and hydrophobic interactions. Glycine residues downstream the selectivity filter are proposed to participate in knob-into-hole contacts with P-helices and S6s. These contacts explain known tetrodotoxin resistance of snakes adapted to toxic prey due to NaV1.4 mutation Ile/Val in the P-helix of repeat 4. Polar residues in P-helices, which are five positions upstream from the selectivity-filter residues, form H-bonds with the ascending-limb backbones. The exceptionally conserved tryptophans are engaged in inter-repeat H-bonds to form a ring whose π-electrons would facilitate the passage of ions from the outer carboxylates to the selectivity filter. The outer-pore model of CaV1.2 derived from the NaV1.4 model is also stabilized by the ring of exceptionally conservative tryptophans and H-bonds between P-helices and ascending limbs. In this model a highly conserved aspartate downstream the selectivity-filter glutamate in repeat II facilitates passage of calcium ions moving to the selectivity-filter ring through the tryptophan ring. Supported by CIHR.

A novel SCN5A mutation associated with the linker between III and IV domains of Nav1.5 in a neonate with fatal long QT syndrome

A male newborn weighing 2334 g was delivered at 37 weeks of gestation by caesarean section because of prenatal ultrasound findings of fetal hydrops with atrioventricualr block, ventriucular tachycardia (VT), and impaired ventricular function. In spite of the intravenous administration of lidocaine, VT continued. He developed poor perfusion and systemic hypotension. After the intravenous administration of amiodarone, VT was terminated. The electrocardiogram revealed an extremely prolonged corrected QT interval (860 ms) with 2:1 atrioventricular block. Unfortunately, he died 18 h after birth in spite of the administration of lidocaine, beta-blocker and magnesium. Mutational analysis identified a novel heterozygous de novo mutation (F1486del) in SCN5A. This mutation is associated with the IFM motif in the linker between III and IV domains of Na(v)1.5, which serves as an inactivation particle binding within the pore of sodium channels. This report demonstrates an interesting relationship between the clinical phenotype and the location of the mutation in long QT syndrome.

Molecular evolution of Na+ channels in teleost fishes

Voltage-dependent sodium channels are critical for electrical excitability. Invertebrates possess a single sodium channel gene; two rounds of genome duplication early in vertebrates increased the number to four. Since the teleost-tetrapod split, independent gene duplications in each lineage have further increased the number of sodium channel genes to 10 in tetrapods and 8 in teleosts. Here we review how the occurrence of multiple sodium channel paralogs has influenced the evolutionary history of three groups of fishes: pufferfish, gymnotiform and mormyriform electric fish. Pufferfish (tetraodontidae) produce a neurotoxin, tetrodotoxin, that binds to and blocks the pore of sodium channels. Pufferfish evolved resistance to their own toxins by amino acid substitutions in the pore of their sodium channels. These substitutions had to occur in parallel across multiple paralogs for organismal resistance to evolve. Gymnotiform and mormyriform fishes independently evolved electric organs to generate electricity for communication and object localization. Two sodium channel genes are expressed in muscle in most fishes. In both groups of weakly electric fishes, one gene lost its expression in muscle and became compartmentalized in the evolutionary novel electric organ, which is a muscle derivative. This gene then evolved at elevated rates, whereas the gene that is still expressed in muscle does not show elevated rates of evolution. In the electric organ-expressing gene, amino acid substitutions occur in parts of the channel involved in determining how long the channel will be open or closed. The enhanced rate of sequence evolution of this gene likely underlies the species-level variations in the electric signal.

<em>In silico</em> modeling of toluene binding site in the pore of voltage-gate sodium channel

In silico modeling of toluene binding site in the pore of voltage-gate sodium channel Thomas RF Scior1, Evelyn Martínez-Morales2, Silvia L Cruz3, Eduardo M Salinas-Stefanon21Departamento de Farmacia; 2Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, Puebla, Pue, México; 3Departamento de Farmacobiología, CINVESTAV-IPN, Calz. Col. Granjas Coapa, MéxicoAbstract: 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, confirming our experimental findings that it inhibits only the WT of skeletal muscle or cardiac isoforms (Nav 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 Nav 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 confirmed the observed toxicity effects.Keywords: Toluene, Na+ channel, Nav1.4 isoform, Nav1.5 isoform, in-silico simulation, cardiotoxicity

The role of stacking interactions in clonidine binding

Stacking interactions of the clonidine aromatic ring with that of Phe or Tyr in the α2-adrenoreceptor and Tyr in the tetrodotoxin-resistant sodium channel pore have been studied. Ab initio quantum-chemical calculations for a model system of two parallel aromatic rings have been performed with the GAMESS software using the 6-31G* basis set in the framework of the second-order Muller-Plesset perturbation theory with full geometry optimization without symmetry constraints. The parallel shifted conformation of two aromatic rings was found to be energetically most favorable. The 2′,6′-chlorination of one of the benzene rings enhances the stacking interaction, somewhat increases the shift of these rings, and possibly improves the hypotensive and analgesic functions of clonidine owing to an increase in the binding energy. The 4-fluorination of the clonidine ring can increase its analgesic effect but practically excludes its hypotensive activity.

Cadmium Trapping in an Epithelial Sodium Channel Pore Mutant

The putative selectivity filter of the epithelial sodium channel (ENaC) comprises a three-residue sequence G/SXS, but it remains uncertain whether the backbone atoms of this sequence or whether their side chains are lining the pore. It has been reported that the S589C mutation in the selectivity filter of alphaENaC renders the channel sensitive to block by externally applied Cd2+; this was interpreted as evidence for Cd2+ coordination with the thiol group of the side chain of alpha589C, pointing toward the pore lumen. Because the alphaS589C mutation alters the monovalent to divalent cation selectivity ratio of ENaC and because internally applied Cd2+ blocks wild-type ENaC with high affinity, we hypothesized that the inhibition of alphaS589C ENaC by Cd2+ results rather from the coordination of this cation with native cysteine residues located in the internal pore of ENaC. We show here that Cd2+ inhibits not only ENaC alphaS589C and alphaS589D but also alphaS589N mutants and that Ca2+ weakly interacts with the S589D mutant. The block of alphaS589C, -D, and -N mutants is characterized by a slow on-rate, is nearly irreversible, is voltage-dependent, and can be prevented by amiloride. The C546S mutation in the second transmembrane helix of gamma subunit in the background of the ENaC alphaS589C, -D, or -N mutants reduces the sensitivity to block by Cd2+ and renders the block rapidly reversible. We conclude therefore that the block by Cd2+ of the alphaS589C, -D, and -N mutants results from the trapping of Cd2+ ions in the internal pore of the channel and involves Cys-546 in the second transmembrane helix of the gammaENaC subunit.

Local anaesthetic block of sodium channels: raising the barrier

Local anaesthetic block of sodium channels: raising the barrier

A sodium channel pore mutation causing Brugada syndrome

Brugada and long QT type 3 syndromes are linked to sodium channel mutations and clinically cause arrhythmias that lead to sudden death. We have identified a novel threonine-to-isoleucine missense mutation at position 353 (T353I) adjacent to the pore-lining region of domain I of the cardiac sodium channel (SCN5A) in a family with Brugada syndrome. Both male and female carriers are symptomatic at young ages, have typical Brugada-type electrocardiogram changes, and have relatively normal corrected QT intervals. To characterize the properties of the newly identified cardiac sodium channel (SCN5A) mutation at the cellular level. Using whole-cell voltage clamp, we found that heterologous expression of SCN5A containing the T353I mutation resulted in 74% +/- 6% less peak macroscopic sodium current when compared with wild-type channels. A construct of the T353I mutant channel fused with green fluorescent protein failed to traffic properly to the sarcolemma, with a large proportion of channels sequestered intracellularly. Overnight exposure to 0.1 mM mexiletine, a Na(+) channel blocking agent, increased T353I channel trafficking to the membrane to near normal levels, but the mutant channels showed a significant late current that was 1.6% +/- 0.2% of peak sodium current at 200 ms, a finding seen with long QT mutations. The clinical presentation of patients carrying the T353I mutation is that of Brugada syndrome and could be explained by a cardiac Na(+) channel trafficking defect. However, when the defect was ameliorated, the mutated channels had biophysical properties consistent with long QT syndrome. The lack of phenotypic changes associated with the long QT syndrome could be explained by a T353I-induced trafficking defect reducing the number of mutant channels with persistent currents present at the sarcolemma.

β1-subunit modulates the Nav1.4 sodium channel by changing the surface charge

Voltage-gated sodium channels, comprised of a pore-forming alpha-subunit and additional regulatory (beta) subunits, play a critical role in regulation of neuronal excitability. Mechanisms of regulation of beta-subunits remain elusive. We have tested the functional effects of beta1 sodium channel subunit on surface charges as a mechanism for channel modulation. HEK-293 cell lines permanently transfected with the sole rat skeletal muscle sodium channel alpha-subunit (Nav1.4), or co-expressing the sodium channel alpha-subunit and beta1-subunit were studied with the whole-cell mode of the patch-clamp technique. At physiological extracellular Ca2+ concentration (2 mM), expression of beta1-subunit did not produce any significant effect on the voltage-dependent properties of sodium currents. However, a shift of half-activation potentials of sodium channel by changing the extracellular Ca2+ was potentiated when beta1 was co-expressed with alpha-subunit. In contrast, the expression of beta1-subunit did not affect the Ca2+ binding to the open or to the closed sodium channel pore, difference of the effect provoked by extracellular Ca2+ could therefore be attributed to an increased in negative surface charge determined by the presence of beta1-subunit. These data are in agreement with the hypothesis of a modulation of the sodium current by the expression of the highly sialylated beta1-subunit, which would alter the channel gating by increasing the density of surface negative charges in the vicinity of the sodium channel voltage sensing machinery.

Minimal sodium channel pore consisting of S5-P-S6 segments preserves intracellular pharmacology

We studied the properties of a sodium channel comprised only of S5-P-S6 region of the rat sodium channel α-subunit Nav1.4 (μ1pore). Results obtained in HEK cell lines permanently transfected with the sodium channel α-subunit or with the μ1pore were compared with data of the native HEK cells. Sodium channel blockers, tetrodotoxin and tetracaine, protect cells transfected with the complete sodium channel against death produced by incubation with veratridine. Veratridine-induced cell death in cell lines expressing the μ1pore construct is antagonised by tetracaine, but not by tetrodotoxin. Whole-cell conductance also increases in the presence of veratridine in μ1pore transfected cells and tetracaine inhibits these currents. Our pharmacological and electrophysiological data suggest that μ1pore keeps binding sites for veratridine and tetracaine, but not for TTX, and reconstitutes the permeation pathway for Na + ions.

SCN9A Mutations Define Primary Erythermalgia as a Neuropathic Disorder of Voltage Gated Sodium Channels

Primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm and painful hands, and/or feet. We previously localized the gene for primary erythermalgia to a 7.94 cM region on chromosome 2q. Recently, Yang et al identified two missense mutations of the sodium channel alpha subunit SCN9A in patients with erythermalgia. The presence of voltage-gated sodium channels in sensory neurons is thought to play a crucial role in several chronic painful neuropathies. We examined four different families and two sporadic cases and detected missense sequence variants in SCN9A to be present in primary erythermalgia patients. A total of five of six mutations were located in highly conserved regions. One family with autosomal dominantly inherited erythermalgia was double heterozygous for two separate SCN9A mutations. These data establish primary erythermalgia as a neuropathic disorder and offers hope for treatment of this incapacitating painful disorder.

Sodium channel mutation leading to saxitoxin resistance in clams increases risk of PSP

Bivalve molluscs, the primary vectors of paralytic shellfish poisoning (PSP) in humans, show marked inter-species variation in their capacity to accumulate PSP toxins (PSTs) which has a neural basis. PSTs cause human fatalities by blocking sodium conductance in nerve fibres. Here we identify a molecular basis for inter-population variation in PSP resistance within a species, consistent with genetic adaptation to PSTs. Softshell clams (Mya arenaria) from areas exposed to 'red tides' are more resistant to PSTs, as demonstrated by whole-nerve assays, and accumulate toxins at greater rates than sensitive clams from unexposed areas. PSTs lead to selective mortality of sensitive clams. Resistance is caused by natural mutation of a single amino acid residue, which causes a 1,000-fold decrease in affinity at the saxitoxin-binding site in the sodium channel pore of resistant, but not sensitive, clams. Thus PSTs might act as potent natural selection agents, leading to greater toxin resistance in clam populations and increased risk of PSP in humans. Furthermore, global expansion of PSP to previously unaffected coastal areas might result in long-term changes to communities and ecosystems.

Tonic and Phasic Guanidinium Toxin-Block of Skeletal Muscle Na Channels Expressed in Mammalian Cells

The blockage of skeletal muscle sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) have been studied in CHO cells permanently expressing rat Nav1.4 channels. Tonic and use-dependent blockage were analyzed in the framework of the ion-trapped model. The tonic affinity (26.6 nM) and the maximum affinity (7.7 nM) of TTX, as well as the "on" and "off" rate constants measured in this preparation, are in remarkably good agreement with those measured for Nav1.2 expressed in frog oocytes, indicating that the structure of the toxin receptor of Nav1.4 and Nav1.2 channels are very similar and that the expression method does not have any influence on the pore properties of the sodium channel. The higher affinity of STX for the sodium channels (tonic and maximum affinity of 1.8 nM and 0.74 nM respectively) is explained as an increase on the "on" rate constant (approximately 0.03 s(-1) nM(-1)), compared to that of TTX (approximately 0.003 s(-1) nM(-1)), while the "off" rate constant is the same for both toxins (approximately 0.02 s(-1)). Estimations of the free-energy differences of the toxin-channel interaction indicate that STX is bound in a more external position than TTX. Similarly, the comparison of the toxins free energy of binding to a ion-free, Na(+)- and Ca(2+)-occupied channel, is consistent with a binding site in the selectivity filter for Ca(2+) more external than for Na(+). This data may be useful in further attempts at sodium-channel pore modeling.

Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block.

Voltage-gated Na+ current is reduced by acid solution. Protons reduce peak Na+ conductance by lowering single channel conductance and shift the voltage range of gating by neutralizing surface charges. Structure-function studies identify six carboxyls and a lysine in the channel's outer vestibule. We examined the roles of the superficial ring of carboxyls in acid block of Na(v)1.4 (the rat skeletal muscle Na+ channel isoform) by measuring the effects of their neutralization or their substitution by lysine on sensitivity to acid solutions, using the two-micropipette voltage clamp in Xenopus oocytes. Alteration of the outer ring of carboxylates had little effect on the voltage for half-activation of Na+ current, as if they are distant from the channels' voltage sensors. The mutations did not abolish proton block; rather, they all shifted the pK(a) (-log of the dissociation constant) in the acid direction. Effects of neutralization on pK(a) were not identical for different mutations, with E758Q > D1241A > D1532N > E403Q. E758K showed double the effect of E758Q, and the other lysine mutations all produced larger effects than the neutralizing mutations. Calculation of the electrostatic potential produced by these carboxylates using a pore model showed that the pK(a) values of carboxylates of Glu-403, Glu-758, and Asp-1532 are shifted to values similar to the experimentally measured pK(a). Calculations also predict the experimentally observed changes in pK(a) that result from mutational neutralization or introduction of a positive charge. We propose that proton block results from partial protonation of these outer ring carboxylates and that all of the carboxylates contribute to a composite Na+ site.

Molecular biology of sodium channels and their role in cardiac arrhythmias

The sodium channel is an integral membrane protein that plays a central role in conduction of the cardiac impulse in working cardiac myocytes and cells of the His-Purkinje system. The channel has two fundamental properties, ion conduction and gating. Specific domains of the channel protein control each of these functions. Ion conduction describes the mechanisms of the selective movement of sodium ion across the pore in the cell membrane. The selectivity of the channel for sodium ions is at least 10 times greater than that for other monovalent cations; the channel does not normally conduct divalent cations. Gating describes the opening and closing of the sodium channel pore. Sodium channels open transiently during membrane depolarization and close by a process termed inactivation. The cardiac sodium channel protein is a multimeric complex consisting of an α and an auxiliary β-subunit. The genes encoding the sodium channel have been cloned and sequenced. The α subunit gene, SCN5A is sufficient to express a functional channel. However, β subunit co-expression increases the level of channel expression and alters the voltage dependence of inactivation. Mutations of the sodium channel may result in incomplete inactivation during maintained depolarization, a decrease in the level of channel expression or acceleration of inactivation. The resulting clinical phenotypes include long QT syndrome, type III (LQT III), Brugada syndrome, and heart block. LQT III and Brugada syndromes have a high case fatality rate and are best treated with an implantable defibrillator.

Epithelial Sodium Channel Pore Region: STRUCTURE AND ROLE IN GATING

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.

A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence.

Voltage-gated sodium (Na(+)) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na(+) channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na(+) channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (I(M); tau approximately 30 ms) and slower (I(S); tau approximately 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the I(M) and I(S) kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na(+) channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

Molecular Dynamics of the Sodium Channel Pore Vary with Gating: Interactions between P-Segment Motions and Inactivation

Disulfide trapping studies have revealed that the pore-lining (P) segments of voltage-dependent sodium channels undergo sizable motions on a subsecond time scale. Such motions of the pore may be necessary for selective ion translocation. Although traditionally viewed as separable properties, gating and permeation are now known to interact extensively in various classes of channels. We have investigated the interaction of pore motions and voltage-dependent gating in micro1 sodium channels engineered to contain two cysteines within the P segments. Rates of catalyzed internal disulfide formation (kSS) were measured in K1237C+W1531C mutant channels expressed in oocytes. During repetitive voltage-clamp depolarizations, increasing the pulse duration had biphasic effects on the kSS, which first increased to a maximum at 200 msec and then decreased with longer depolarizations. This result suggested that occupancy of an intermediate inactivation state (IM) facilitates pore motions. Consistent with the known antagonism between alkali metals and a component of slow inactivation, kSS varied inversely with external [Na+]o. We examined the converse relationship, namely the effect of pore flexibility on gating, by measuring recovery from inactivation in Y401C+E758C (YC/EC) channels. Under oxidative conditions, recovery from inactivation was slower than in a reduced environment in which the spontaneous YC/EC cross-link is disrupted. The most prominent effects were slowing of a component with intermediate recovery kinetics, with diminution of its relative amplitude. We conclude that occupancy of an intermediate inactivation state facilitates motions of the P segments; conversely, flexibility of the P segments alters an intermediate component of inactivation.

Structure of the sodium channel pore deduced from interaction with local anesthetic antiarrhythmic drugs.

Structure of the sodium channel pore deduced from interaction with local anesthetic antiarrhythmic drugs.