muO conotoxins inhibit NaV channels by interfering with their voltage sensors in domain-2

The muO-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (Na(V)1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which muO-conotoxins affect Na(V) channels. Rat Na(V)1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily mu-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the Na(V) channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion beta-toxins. In fact, MrVIA functionally competed with the scorpion beta-toxin Ts1 from Tityus serrulatus, while it did not show competition with mu-GIIIA. Ts1 and mu-GIIIA did not compete either. Thus, similar to scorpion beta-toxins, muO-conotoxins are voltage-sensor toxins targeting receptor site-4 on Na(V) channels. They "block" Na(+) flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.     

Conotoxin modulation of voltage-gated sodium channels

The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the muO-conotoxins which inhibit VGSC Na(+) conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the muO-conotoxins, which may develop into potential drug leads for the treatment of pain states.     

Structure/function characterization of micro-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. Micro-conotoxin KIIIA is representative of micro-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only approximately 20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both Na(V)1.2 and Na(V)1.6 were strongly blocked; within experimental wash times of 40-60 min, block was reversed very little for Na(V)1.2 and only partially for Na(V)1.6. Other isoforms were blocked reversibly: Na(V)1.3 (IC50 8 microM), Na(V)1.5 (IC50 284 microM), and Na(V)1.4 (IC50 80 nM). "Alanine-walk" and related analogs were synthesized and tested against both Na(V)1.2 and Na(V)1.4; replacement of Trp-8 resulted in reversible block of Na(V)1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na(V)1.4 than of Na(V)1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na(V)1.2 and that further engineering of micro-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.     

Scorpion β-toxin interference with NaV channel voltage sensor gives rise to excitatory and depressant modes

Scorpion β toxins, peptides of ～70 residues, specifically target voltage-gated sodium (Na(V)) channels to cause use-dependent subthreshold channel openings via a voltage-sensor trapping mechanism. This excitatory action is often overlaid by a not yet understood depressant mode in which Na(V) channel activity is inhibited. Here, we analyzed these two modes of gating modification by β-toxin Tz1 from Tityus zulianus on heterologously expressed Na(V)1.4 and Na(V)1.5 channels using the whole cell patch-clamp method. Tz1 facilitated the opening of Na(V)1.4 in a use-dependent manner and inhibited channel opening with a reversed use dependence. In contrast, the opening of Na(V)1.5 was exclusively inhibited without noticeable use dependence. Using chimeras of Na(V)1.4 and Na(V)1.5 channels, we demonstrated that gating modification by Tz1 depends on the specific structure of the voltage sensor in domain 2. Although residue G658 in Na(V)1.4 promotes the use-dependent transitions between Tz1 modification phenotypes, the equivalent residue in Na(V)1.5, N803, abolishes them. Gating charge neutralizations in the Na(V)1.4 domain 2 voltage sensor identified arginine residues at positions 663 and 669 as crucial for the outward and inward movement of this sensor, respectively. Our data support a model in which Tz1 can stabilize two conformations of the domain 2 voltage sensor: a preactivated outward position leading to Na(V) channels that open at subthreshold potentials, and a deactivated inward position preventing channels from opening. The results are best explained by a two-state voltage-sensor trapping model in that bound scorpion β toxin slows the activation as well as the deactivation kinetics of the voltage sensor in domain 2.     

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.     

Anticonvulsant activity of 2,4(1H)-diarylimidazoles in mice and rats acute seizure models

2,4(1H)-Diarylimidazoles have been previously shown to inhibit hNa(V)1.2 sodium (Na) channel currents. Since many of the clinically used anticonvulsants are known to inhibit Na channels as an important mechanism of their action, these compounds were tested in two acute rodent seizure models for anticonvulsant activity (MES and scMet) and for sedative and ataxic side effects. Compounds exhibiting antiepileptic activity were further tested to establish a dose response curve (ED(50)). The experimental data identified four compounds with anticonvulsant activity in the MES acute seizure rodent model (compound 10, ED(50)=61.7mg/kg; compound 13, ED(50)=46.8mg/kg, compound 17, ED(50)=129.5mg/kg and compound 20, ED(50)=136.7mg/kg). Protective indexes (PI=TD(50)/ED(50)) ranged from 2.1 (compound 10) to greater than 3.6 (compounds 13, 17 and 20). All four compounds were shown to inhibit hNa(V)1.2 in a dose dependant manner. Even if a correlation between sodium channel inhibition and anticonvulsant activity was unclear, these studies identify four Na channel antagonists with anticonvulsant activity, providing evidence that these derivatives could be potential drug candidates for development as safe, new and effective antiepileptic drugs (AEDs).     

Calmodulin regulation of Nav1.4 current: role of binding to the carboxyl terminus

Calmodulin (CaM) regulates steady-state inactivation of sodium currents (Na(V)1.4) in skeletal muscle. Defects in Na current inactivation are associated with pathological muscle conditions such as myotonia and paralysis. The mechanisms of CaM modulation of expression and function of the Na channel are incompletely understood. A physical association between CaM and the intact C terminus of Na(V)1.4 has not previously been demonstrated. FRET reveals channel conformation-independent association of CaM with the C terminus of Na(V)1.4 (CT-Na(V)1.4) in mammalian cells. Mutation of the Na(V)1.4 CaM-binding IQ motif (Na(V)1.4(IQ/AA)) reduces cell surface expression of Na(V)1.4 channels and eliminates CaM modulation of gating. Truncations of the CT that include the IQ region abolish Na current. Na(V)1.4 channels with one CaM fused to the CT by variable length glycine linkers exhibit CaM modulation of gating only with linker lengths that allowed CaM to reach IQ region. Thus one CaM is sufficient to modulate Na current, and CaM acts as an ancillary subunit of Na(V)1.4 channels that binds to the CT in a conformation-independent fashion, modulating the voltage dependence of inactivation and facilitating trafficking to the surface membrane.     

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.     

Investigation of the relationship between the structure and function of Ts2, a neurotoxin from Tityus serrulatus venom

Scorpion toxins targeting voltage-gated sodium (Na(V)) channels are peptides that comprise 60-76 amino acid residues cross-linked by four disulfide bridges. These toxins can be divided in two groups (α and β toxins), according to their binding properties and mode of action. The scorpion α-toxin Ts2, previously described as a β-toxin, was purified from the venom of Tityus serrulatus, the most dangerous Brazilian scorpion. In this study, seven mammalian Na(V) channel isoforms (rNa(V)1.2, rNa(V)1.3, rNa(V)1.4, hNa(V)1.5, mNa(V)1.6, rNa(V)1.7 and rNa(V)1.8) and one insect Na(V) channel isoform (DmNa(V)1) were used to investigate the subtype specificity and selectivity of Ts2. The electrophysiology assays showed that Ts2 inhibits rapid inactivation of Na(V)1.2, Na(V)1.3, Na(V)1.5, Na(V)1.6 and Na(V)1.7, but does not affect Na(V)1.4, Na(V)1.8 or DmNa(V)1. Interestingly, Ts2 significantly shifts the voltage dependence of activation of Na(V)1.3 channels. The 3D structure of this toxin was modeled based on the high sequence identity (72%) shared with Ts1, another T. serrulatus toxin. The overall fold of the Ts2 model consists of three β-strands and one α-helix, and is arranged in a triangular shape forming a cysteine-stabilized α-helix/β-sheet (CSαβ) motif.     

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.     

Local anesthetic inhibition of a bacterial sodium channel

Recent structural breakthroughs with the voltage-gated sodium channel from Arcobacter butzleri suggest that such bacterial channels may provide a structural platform to advance the understanding of eukaryotic sodium channel gating and pharmacology. We therefore set out to determine whether compounds known to interact with eukaryotic Na(V)s could also inhibit the bacterial channel from Bacillus halodurans and NaChBac and whether they did so through similar mechanisms as in their eukaryotic homologues. The data show that the archetypal local anesthetic (LA) lidocaine inhibits resting NaChBac channels with a dissociation constant (K(d)) of 260 μM, and channels displayed a left-shifted steady-state inactivation gating relationship in the presence of the drug. Extracellular application of QX-314 to expressed NaChBac channels had no effect on sodium current, whereas internal exposure via injection of a bolus of the quaternary derivative rapidly reduced sodium conductance, consistent with a hydrophilic cytoplasmic access pathway to an internal binding site. However, the neutral derivative benzocaine applied externally inhibited NaChBac channels, suggesting that hydrophobic pathways can also provide drug access to inhibit channels. Alternatively, ranolazine, a putative preopen state blocker of eukaryotic Na(V)s, displayed a K(d) of 60 μM and left-shifted the NaChBac activation-voltage relationship. In each case, block enhanced entry into the inactivated state of the channel, an effect that is well described by a simple kinetic scheme. The data suggest that although significant differences exist, LA block of eukaryotic Na(V)s also occurs in bacterial sodium channels and that NaChBac shares pharmacological homology to the resting state of vertebrate Na(V) homologues.     

Structure, dynamics, and selectivity of the sodium channel blocker mu-conotoxin SIIIA

mu-SIIIA, a novel mu-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of mu-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, mu-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11-16, while residues 3-5 in the N-terminal region form a helix-like turn resembling 3 10-helix. The Trp12 and His16 side chains are close together, as in the related conotoxin mu-SmIIIA, but Asn2 is more distant. Dynamics measurements show that the N-terminus and Ser9 have larger-magnitude motions on the subnanosecond time scale, while the C-terminus is more rigid. Cys4, Trp12, and Cys13 undergo significant conformational exchange on microsecond to millisecond time scales. mu-SIIIA is a potent, nearly irreversible blocker of Na V1.2 but also blocks Na V1.4 and Na V1.6 with submicromolar potency. The selectivity profile of mu-SIIIA, including poor activity against the cardiac sodium channel, Na V1.5, is similar to that of the closely related mu-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal Na V1.2. The structural and functional characterization described in this paper of an analgesic mu-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.     

The cation selectivity filter of the bacterial sodium channel,NaChBac

The Bacillus halodurans voltage-gated sodium-selective channel (NaChBac) (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001b. SCIENCE: 294:2372-2375), is an ideal candidate for high resolution structural studies because it can be expressed in mammalian cells and its functional properties studied in detail. It has the added advantage of being a single six transmembrane (6TM) orthologue of a single repeat of mammalian voltage-gated Ca(2+) (Ca(V)) and Na(+) (Na(V)) channels. Here we report that six amino acids in the pore domain (LESWAS) participate in the selectivity filter. Replacing the amino acid residues adjacent to glutamatic acid (E) by a negatively charged aspartate (D; LEDWAS) converted the Na(+)-selective NaChBac to a Ca(2+)- and Na(+)-permeant channel. When additional aspartates were incorporated (LDDWAD), the mutant channel resulted in a highly expressing voltage-gated Ca(2+)-selective conductance.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Delta-conotoxin structure/function through a cladistic analysis.

Delta-conotoxins are Conus peptides that inhibit inactivation of voltage-gated sodium channels. The suggestion that delta-conotoxins might be an essential component of the venoms of fish-hunting cone snails which rapidly immobilize their prey [Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151] has not been tested. On the basis of cDNA cloning, all of the fish-hunting Conus analyzed yielded at least one delta-conotoxin sequence. In addition, one delta-conotoxin isolated from the venom of Conus striatus had an amino acid sequence identical to that predicted from cDNA cloning. This new peptide exhibited properties of delta-conotoxins: it targeted sodium channels and potentiated action potentials by slowing channel inactivation. Homologous sequences of delta-conotoxins from two groups (clades) of related fish-hunting Conus species share consensus features but differ significantly from the two known delta-conotoxins from mollusc-hunting Conus venoms. Three large hydrophobic amino acids were conserved; analogues of the previously described delta-conotoxin PVIA with alanine substituted for the conserved amino acids F9 and I12 lost substantial biological activity. In contrast, both the T8A and K13A delta-conotoxin PVIA analogues, where substitutions were at nonconserved loci, proved to be biologically active. Taken together, our results indicate that a cladistic approach can identify amino acids critical for the activity of conotoxins and provide extensive information as to which amino acid substitutions can be made without significant functional consequences.     

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 alpha-subunit Nav1.4 (micro1pore). Results obtained in HEK cell lines permanently transfected with the sodium channel alpha-subunit or with the micro1pore 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 micro1pore construct is antagonised by tetracaine, but not by tetrodotoxin. Whole-cell conductance also increases in the presence of veratridine in micro1pore transfected cells and tetracaine inhibits these currents. Our pharmacological and electrophysiological data suggest that micro1pore keeps binding sites for veratridine and tetracaine, but not for TTX, and reconstitutes the permeation pathway for Na+ ions.     

Altered expression and function of sodium channels in large DRG neurons and myelinated A-fibers in early diabetic neuropathy in the rat

Differential alterations of sodium channels in small nociceptive C-fiber DRG neurons have been implicated in diabetic neuropathy. In this study, we investigated sodium currents and the expression of sodium channels in large A-fiber DRG neurons in diabetic rats. Compared with controls, large neurons from diabetic rats showed significant increases in both total and TTX-S sodium currents and approximately -15mV shifts in their voltage-dependent activation kinetics. TTX-R Na(v)1.9 sodium current was also significantly increased, whereas no alteration of TTX-R Na(v)1.8 current was observed in neurons from diabetic rats. Sodium current induced by fast- or slow-voltage ramps increased markedly in the diabetic neurons as well. Immunofluorescence studies showed significant increases in the levels and number of large DRG neurons from diabetic rats expressing Na(v)1.2, Na(v)1.3, Na(v)1.7, and Na(v)1.9 whereas Na(v)1.8 decreased. We also observed a decrease in the number of nodes of Ranvier expressing Na(v)1.8 and in staining intensity of Na(v)1.6 and Na(v)1.8 at nodes. Our results suggest that alterations of sodium channels occur in large DRG neurons and A-fibers, and may play an important role in diabetic sensory neuropathy.     

Ion permeation through a voltage- sensitive gating pore in brain sodium channels having voltage sensor mutations

Voltage-gated sodium channels activate in response to depolarization, but it is unknown whether the voltage-sensing arginines in their S4 segments pivot across the lipid bilayer as voltage sensor paddles or move through the protein in a gating pore. Here we report that mutation of pairs of arginine gating charges to glutamine induces cation permeation through a gating pore in domain II of the Na(V)1.2a channel. Mutation of R850 and R853 induces a K(+)-selective inward cationic current in the resting state that is blocked by activation. Remarkably, mutation of R853 and R856 causes an outward cationic current with the opposite gating polarity. These results support a model in which the IIS4 gating charges move through a narrow constriction in a gating pore in the sodium channel protein during gating. Paired substitutions of glutamine allow cation movement through the constriction when appropriately positioned by the gating movements of the S4 segment.     

Ion solvation and structural stability in a sodium channel investigated by molecular dynamics calculations

The stability and ion binding properties of the homo-tetrameric pore domain of a prokaryotic, voltage-gated sodium channel are studied by extensive all-atom molecular dynamics simulations, with the channel protein being embedded in a fully hydrated lipid bilayer. It is found that Na(+) ion presents in a mostly hydrated state inside the wide pore of the selectivity filter of the sodium channel, in sharp contrast to the nearly fully dehydrated state for K(+) ions in potassium channels. Our results also indicate that Na(+) ions make contact with only one or two out of the four polypeptide chains forming the selectivity filter, and surprisingly, the selectivity filter exhibits robust stability for various initial ion configurations even in the absence of ions. These findings are quite different from those in potassium channels. Furthermore, an electric field above 0.5V/nm is suggested to be able to induce Na(+) permeation through the selectivity filter.