Native Pyroglutamation of Huwentoxin-IV: A Post-Translational Modification that Increases the Trapping Ability to the Sodium Channel

Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC₅₀ nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.     

Functional Analysis of the Kv1.1 N255D Mutation Associated with Autosomal Dominant Hypomagnesemia

Mutations in the voltage-gated K⁺ channel Kv1.1 have been linked with a mixed phenotype of episodic ataxia and/or myokymia. Recently, we presented autosomal dominant hypomagnesemia as a new phenotypic characteristic associated with a mutation in Kv1.1 (N255D) (Glaudemans, B., van der Wijst, J., Scola, R. H., Lorenzoni, P. J., Heister, A., van der Kemp, A. W., Knoers, N. V., Hoenderop, J. G., and Bindels, R. J. (2009) J. Clin. Invest. 119, 936–942). A conserved asparagine at position 255 in the third transmembrane segment was converted into an aspartic acid, resulting in a non-functional channel. In this study, we explored the functional consequence of this conserved residue by substitution with other hydrophobic, polar, or charged amino acids (N255E, N255Q, N255A, N255V, N255T, and N255H). Upon overexpression in human embryonic kidney (HEK293) cells, cell surface biotinylation revealed plasma membrane expression of all mutant channels. Next, we used the whole-cell patch clamp technique to demonstrate that the N255E and N255Q mutants were non-functional. Substitution of Asn-255 with other amino acids (N255A, N255V, N255T, and N255H) did not prevent ion conduction, and these mutant channels activated at more negative potentials when compared with wild-type channels, −41.5 ± 1.6, −45.5 ± 2.0, −50.5 ± 1.9, and −33.8 ± 1.3 mV to −29.4 ± 1.1 mV, respectively. The time constant of activation was significantly faster for the two most hydrophobic mutations, N255A (6.2 ± 0.2 ms) and N255V (5.2 ± 0.3 ms), and the hydrophilic mutant N255T (9.8 ± 0.4 ms) in comparison with wild type (13.0 ± 0.9 ms). Furthermore, the voltage dependence of inactivation was shifted ∼13 mV to more negative potentials in all mutant channels except for N255H. Taken together, our data showed that an asparagine at position 255 in Kv1.1 is required for normal voltage dependence and kinetics of channel gating.     

Genetic basis of tetrodotoxin resistance in pufferfishes

Tetrodotoxin (TTX) is a highly potent neurotoxin that selectively binds to the outer vestibule of voltage-gated sodium channels. Pufferfishes accumulate extremely high concentrations of TTX without any adverse effect. A nonaromatic amino acid (Asn) residue present in domain I of the pufferfish, Takifugu pardalis, Na v1.4 channel has been implicated in the TTX resistance of pufferfishes . However, the effect of this residue on TTX sensitivity has not been investigated, and it is not known if this residue is conserved in all pufferfishes. We have investigated the genetic basis of TTX resistance in pufferfishes by comparing the sodium channels from two pufferfishes (Takifugu rubripes [fugu] and Tetraodon nigroviridis) and the TTX-sensitive zebrafish. Although all three fishes contain duplicate copies of Na v1.4 channels (Na v1.4a and Na v1.4b), several substitutions were found in the TTX binding outer vestibule of the two pufferfish channels. Electrophysiological studies showed that the nonaromatic residue (Asn in fugu and Cys in Tetraodon) in domain I of Na v1.4a channels confers TTX resistance. The Glu-to-Asp mutation in domain II of Tetraodon channel Na v1.4b is similar to that in the saxitoxin- and TTX-resistant Na+ channels of softshell clams . Besides helping to deter predators, TTX resistance enables pufferfishes to selectively feed on TTX-bearing organisms.     

Sodium Channelopathies: From Molecular Physiology towards Medical Genetics

Voltage-gated sodium channels are heteromeric transmembrane proteins involved in the conduction of sodium ion currents in response to membrane depolarization. In humans, nine homologous genes, SCN1A–11A, which encode different isoforms of the voltage-gated sodium channel family, are known. Sodium channel isoforms exhibit different kinetic properties that determine different types of neurons. Mutations in different channels are described in patients with various congenital disorders, from epilepsy to congenital insensitivity to pain. This review presents an analysis of the current literature on the properties of different isoforms of voltage-gated sodium channels and associated diseases.     

Sodium ionic and gating currents in mammalian cells

Ionic and gating currents from voltage-gated sodium channels were recorded in mouse neuroblastoma cells using the path-clamp technique. Displacement currents were measured from whole-cell recordings. The gating charge displaced during step depolarizations increased with the applied membrane potential and reached saturating levels above 20 mV Prolonged large depolarizations produced partial immobilization of the gating charge, and only about one third of the displaced charge was quickly reversed upon return to negative holding potentials. The activation and inactivation properties of macroscopic sodium currents were characterized by voltage-clamp analysis of large outside-out patches and the single-channel conductance was estimated from nonstationary noise analysis. The general properties of the sodium channels in mouse neuroblastoma cells are very similar to those previously reported for various preparations of invertebrate and vertebrate nerve cells. Offprint requests to: O. Moran     

Mechanism of tetrodotoxin block and resistance in sodium channels

Tetrodotoxin (TTX) has been used for many decades to characterize the structure and function of biological ion channels. Yet, the precise mechanism by which TTX blocks voltage-gated sodium (Na_V) channels is not fully understood. Here molecular dynamics simulations are used to elucidate how TTX blocks mammalian voltage-gated sodium (Nav) channels and why it fails to be effective for the bacterial sodium channel, Na_VAb. We find that, in Na_VAb, a sodium ion competes with TTX for the binding site at the extracellular end of the filter, thus reducing the blocking efficacy of TTX. Using a model of the skeletal muscle channel, Na_V1.4, we show that the conduction properties of the channel observed experimentally are faithfully reproduced. We find that TTX occludes the entrance of Na_V1.4 by forming a network of hydrogen-bonds at the outer lumen of the selectivity filter. The guanidine group of TTX adopts a lateral orientation, rather than pointing into the filter as proposed previously. The acidic residues just above the selectivity filter are important in stabilizing the hydrogen-bond network between TTX and Na_V1.4. The effect of two single mutations of a critical tyrosine residue in the filter of Na_V1.4 on TTX binding observed experimentally is reproduced using computational mutagenesis.     

Splice Variants of Na(V)1.7 Sodium Channels Have Distinct β Subunit-Dependent Biophysical Properties

Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na_V1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na_V1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na_V1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027), the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012). The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na_V channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.     

T-type Channels Become Highly Permeable to Sodium Ions Using an Alternative Extracellular Turret Region (S5-P) Outside the Selectivity Filter

T-type (Ca_v3) channels are categorized as calcium channels, but invertebrate ones can be highly sodium-selective channels. We illustrate that the snail LCa_v3 T-type channel becomes highly sodium-permeable through exon splicing of an extracellular turret and descending helix in domain II of the four-domain Ca_v3 channel. Highly sodium-permeable T-type channels are generated without altering the invariant ring of charged residues in the selectivity filter that governs calcium selectivity in calcium channels. The highly sodium-permeant T-type channel expresses in the brain and is the only splice isoform expressed in the snail heart. This unique splicing of turret residues offers T-type channels a capacity to serve as a pacemaking sodium current in the primitive heart and brain in lieu of Na_v1-type sodium channels and to substitute for voltage-gated sodium channels lacking in many invertebrates. T-type channels would also contribute substantially to sodium leak conductances at rest in invertebrates because of their large window currents.     

Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei

Background

Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear.

Methods/Principal Findings

We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P _K+/P _Cl−∼0.31), while the other two types of channels are slightly selective for cations (P _K+/P _Cl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel''s pore.

Conclusions/Significance

These results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.     

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl⁻ concentrations in the kidney and in other epithelia. We tested whether Cl⁻ alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl⁻ with SO₄²⁻, H₂PO₄⁻, or SCN⁻ produced a large increase in ENaC current, indicating that extracellular Cl⁻ inhibits ENaC. Extracellular Cl⁻ also inhibited ENaC in Na⁺-transporting epithelia. The anion selectivity sequence was SCN⁻ < SO₄²⁻ < H₂PO₄⁻ < F⁻ < I⁻ < Cl⁻ < Br⁻. Crystallization of ASIC1a revealed a Cl⁻ binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (α_H418A and β_R388A) disrupted the response to Cl⁻, suggesting that Cl⁻ might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl⁻. The response to Cl⁻ was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl⁻. Cl⁻ regulated ENaC activity in part through enhanced Na⁺ self-inhibition, a process by which extracellular Na⁺ inhibits ENaC. Together, the data indicate that extracellular Cl⁻ regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl⁻ might modulate epithelial Na⁺ absorption.The epithelial Na⁺ channel ENaC² is a heterotrimer of homologous α, β, and γ subunits (1, 2). ENaC functions as a pathway for Na⁺ absorption across epithelial cells in the kidney collecting duct, lung, distal colon, and sweat duct (reviewed in Refs. 3 and 4). Na⁺ transport is critical for the maintenance of Na⁺ homeostasis and for the control of the composition and quantity of the fluid on the apical membrane of these epithelia. ENaC mutations and defects in its regulation cause inherited forms of hypertension and hypotension (5) and may contribute to the pathogenesis of lung disease in cystic fibrosis (6).ENaC is a member of the DEG/ENaC family of ion channels. A common structural feature of these channels is a large extracellular domain that plays a critical role in channel gating. For example, in ASICs, the extracellular domain functions as a receptor for protons, which transiently activate the channel by titrating residues that form an acidic pocket (7). FaNaCh is a ligand-gated family member in Helix aspersa, activated by the peptide FMRFamide (8). In Caenorhabditis elegans MEC family members, the extracellular domain is thought to respond to mechanical signals (9).ENaC differs from other family members because it is constitutively active in the absence of a ligand/stimulus. However, a convergence of data indicate that ENaC gating is modulated by a variety of molecules that bind to or modify its extracellular domains, including proteases (10–12), Na⁺ (13–15), protons (16), and the divalent cations Zn²⁺ and Ni²⁺ (17, 18). These findings suggest that the ENaC extracellular domain might regulate epithelial Na⁺ transport by sensing and integrating diverse signals in the extracellular environment.In the current study, we tested the hypothesis that ENaC activity is regulated by changes in the extracellular Cl⁻ concentration. Several observations suggested that Cl⁻ might be a strong candidate to regulate the channel. First, transport of Na⁺ and Cl⁻ are often coupled to maintain electroneutrality. Second, ENaC is exposed to large changes in extracellular Cl⁻ concentration. For example, in the kidney collecting duct, the urine Cl⁻ concentration varies widely (19). As the predominant anion, its concentration parallels that of Na⁺ in most clinical states. However, under conditions of metabolic alkalosis and metabolic acidosis, the Na⁺ and Cl⁻ concentrations can become dissociated as a result of increased urinary bicarbonate (alkalosis) or ammonium (acidosis) (19). Thus, ENaC is well positioned to respond to changes in Cl⁻ concentration. Third, crystallization of ASIC1a revealed a binding site for a Cl⁻ ion at the base of the thumb domain (7). The Cl⁻ is coordinated by Arg-310 and Glu-314 from one subunit and Lys-212 from an adjacent subunit. Although the functional role of Cl⁻ binding to ASIC1a is unknown, it supports the hypothesis that extracellular Cl⁻ might regulate the activity of DEG/ENaC ion channels.     

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.     

Developmental Appearance of Sodium Channel Subtypes in Rat Skeletal Muscle Cultures

22Na influx was measured in the established muscle cell line L-6 and in primary rat skeletal muscle cultures following activation of sodium channels by veratridine and sea anemone toxin II. Inhibition of the activated channels by tetrodotoxin (TTX) was analyzed with computer-assisted fits to one- or two-site binding models. In L-6 cultures, two inhibitable sodium channel populations were resolved at all ages in culture: a TTX-sensitive (K = 0.6-5.0 X 10(-8) M) and an insensitive population (Ki = 3.3-4.9 X 10(-6) M). In primary rat muscle cultures, the sensitivity of the toxin-stimulated channels to TTX changed with time in culture. In 4-day-old cultures, a single sodium channel population was detected using TTX (Ki = 2.4 X 10(-7)M). A single population was also found in 6-day-old cultures (Ki = 5.3 X 10(-7) M). By day 7 in culture, the inhibition of 22Na influx by TTX could be resolved into two components with high- and low-affinity sites for the toxin (Ki = 1.3 X 10(-9) M and 9.6 X 10(-7) M). We conclude that a single, toxin-activated sodium channel population with low affinity for TTX exists at early stages, whereas a second, high-affinity population evolves with time in primary rat muscle cultures. The expression of a high-affinity site apparently does not require ongoing neuronal involvement and may reflect an intrinsic property of the muscle cells.     

Structure of a putative sodium channel from the sea anemoneAiptasia pallida

A cDNA encoding a full length putative sodium channel has been cloned from the sea anemoneAiptasia pallida. The deduced protein, named AiNal, has a predicted molecular weight of 205 000 Da. It shows high structural similarity to other sodium channels from both invertebrates and vertebrates, and its structure is consistent with the four domain, six transmembrane segment motif of all known voltage-gated sodium channels. In the region purported to constitute the tetrodotoxin (TTX) receptor of sodium channels, AiNal differs from the TTX-sensitive motif, suggesting that currents carried by this channel would be insensitive to TTX. The presence of a conventional sodium channel protein in anemones indicates, for the first time, that neurons in sea anemones are likely to be capable of producing fast, overshooting action, potentials.     

Sodium channel activation underlies transfluthrin repellency in Aedes aegypti

BackgroundVolatile pyrethroid insecticides, such as transfluthrin, have received increasing attention for their potent repellent activities in recent years for controlling human disease vectors. It has been long understood that pyrethroids kill insects by promoting activation and inhibiting inactivation of voltage-gated sodium channels. However, the mechanism of pyrethroid repellency remains poorly understood and controversial.Methodology/Principal findingsHere, we show that transfluthrin repels Aedes aegypti in a hand-in-cage assay at nonlethal concentrations as low as 1 ppm. Contrary to a previous report, transfluthrin does not elicit any electroantennogram (EAG) responses, indicating that it does not activate olfactory receptor neurons (ORNs). The 1S-cis isomer of transfluthrin, which does not activate sodium channels, does not elicit repellency. Mutations in the sodium channel gene that reduce the potency of transfluthrin on sodium channels decrease transfluthrin repellency but do not affect repellency by DEET. Furthermore, transfluthrin enhances DEET repellency.Conclusions/SignificanceThese results provide a surprising example that sodium channel activation alone is sufficient to potently repel mosquitoes. Our findings of sodium channel activation as the principal mechanism of transfluthrin repellency and potentiation of DEET repellency have broad implications in future development of a new generation of dual-target repellent formulations to more effectively repel a variety of human disease vectors.     

Electrophysiological characterization of Tityus obscurus β toxin 1 (To1) on Na+-channel isoforms

To1, previously named Tc49b, is a peptide neurotoxin isolated from venom of the scorpion Tityus obscurus that is responsible for lethal human poisoning cases in the Brazilian Amazonian region. Previously, To1 was shown to be lethal to mice and to change Na⁺ permeation in cerebellum granular neurons from rat brain. In addition, To1 did not affect Shaker B K⁺ channels. Based on sequence similarities, To1 was described as a β-toxin. In the present work, To1 was purified from T. obscurus venom and submitted to an electrophysiological characterization in human and invertebrate Na_V channels. The analysis of the electrophysiological experiments reveal that To1 enhances the open probability at more negative potentials of human Na_V 1.3 and 1.6, of the insect channel BgNa_V1 and of arachnid VdNa_V1 channel. In addition, To1 reduces the peak of Na⁺ currents in some of the Na_Vs tested. These results support the classification of the To1 as a β-toxin. A structure and functional comparison to other β-toxins that share sequence similarity to To1 is also presented.     

The effects of huwentoxin-I on the voltage-gated sodium channels of rat hippocampal and cockroach dorsal unpaired median neurons

Huwentoxin-I (HWTX-I) is a 33-residue peptide isolated from the venom of Ornithoctonus huwena and could inhibit TTX-sensitive voltage-gated sodium channels and N-type calcium channels in mammalian dorsal root ganglion (DRG) neurons. However, the effects of HWTX-I on mammalian central neuronal and insect sodium channel subtypes remain unknown. In this study, we found that HWTX-I potently inhibited sodium channels in rat hippocampal and cockroach dorsal unpaired median (DUM) neurons with the IC₅₀ values of 66.1 ± 5.2 and 4.80 ± 0.58 nM, respectively. Taken together with our previous work on DRG neurons (IC₅₀ ≈ 55 nM), the order of sodium channel sensitivity to HWTX-I inhibition was insect central DUM ? mammalian peripheral > mammalian central neurons. HWTX-I exhibited no effect on the steady-state activation and inactivation of sodium channels in rat hippocampal and cockroach DUM neurons.