Historical Perspective of the Characterization of Conotoxins Targeting Voltage-Gated Sodium Channels.

Marine toxins have potent actions on diverse sodium ion channels regulated by transmembrane voltage (voltage-gated ion channels) or by neurotransmitters (nicotinic acetylcholine receptor channels). Studies of these toxins have focused on varied aspects of venom peptides ranging from evolutionary relationships of predator and prey, biological actions on excitable tissues, potential application as pharmacological intervention in disease therapy, and as part of multiple experimental approaches towards an understanding of the atomistic characterization of ion channel structure. This review examines the historical perspective of the study of conotoxin peptides active on sodium channels gated by transmembrane voltage, which has led to recent advances in ion channel research made possible with the exploitation of the diversity of these marine toxins.

Charge substitutions at the voltage-sensing module of domain III enhance actions of site-3 and site-4 toxins on an insect sodium channel.

Scorpion α-toxins bind at the pharmacologically-defined site-3 on the sodium channel and inhibit channel inactivation by preventing the outward movement of the voltage sensor in domain IV (IVS4), whereas scorpion ß-toxins bind at site-4 on the sodium channel and enhance channel activation by trapping the voltage sensor of domain II (IIS4) in its outward position. However, limited information is available on the role of the voltage-sensing modules (VSM, comprising S1-S4) of domains I and III in toxin actions. We have previously shown that charge reversing substitutions of the innermost positively-charged residues in IIIS4 (R4E, R5E) increase the activity of an insect-selective site-4 scorpion toxin, Lqh-dprIT3-c, on BgNav1-1a, a cockroach sodium channel. Here we show that substitutions R4E and R5E in IIIS4 also increase the activity of two site-3 toxins, LqhαIT from Leiurusquinquestriatus hebraeus and insect-selective Av3 from Anemonia viridis. Furthermore, charge reversal of either of two conserved negatively-charged residues, D1K and E2K, in IIIS2 also increase the action of the site-3 and site-4 toxins. Homology modeling suggests that S2-D1 and S2-E2 interact with S4-R4 and S4-R5 in the VSM of domain III (III-VSM), respectively, in the activated state of the channel. However, charge swapping between S2-D1 and S4-R4 had no compensatory effects on gating or toxin actions, suggesting that charged residue interactions are complex. Collectively, our results highlight the involvement of III-VSM in the actions of both site 3 and site 4 toxins, suggesting that charge reversing substitutions in III-VSM allosterically facilitate IIS4 or IVS4 voltage sensor trapping by these toxins.

Roles for Countercharge in the Voltage Sensor Domain of Ion Channels.

Voltage-gated ion channels share a common structure typified by peripheral, voltage sensor domains. Their S4 segments respond to alteration in membrane potential with translocation coupled to ion permeation through a central pore domain. The mechanisms of gating in these channels have been intensely studied using pioneering methods such as measurement of charge displacement across a membrane, sequencing of genes coding for voltage-gated ion channels, and the development of all-atom molecular dynamics simulations using structural information from prokaryotic and eukaryotic channel proteins. One aspect of this work has been the description of the role of conserved negative countercharges in S1, S2, and S3 transmembrane segments to promote sequential salt-bridge formation with positively charged residues in S4 segments. These interactions facilitate S4 translocation through the lipid bilayer. In this review, we describe functional and computational work investigating the role of these countercharges in S4 translocation, voltage sensor domain hydration, and in diseases resulting from countercharge mutations.

Ribbon α-Conotoxin KTM Exhibits Potent Inhibition of Nicotinic Acetylcholine Receptors.

KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed α-conotoxin (α-CTx) that demonstrates potent inhibition of rat α3ß2-nicotinic acetylcholine receptors (rα3ß2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of rα3ß2-nAChRs by KTM (IC50 = 0.19 ± 0.02 nM), and inhibition of the same nAChR isoform by α-CTx MII (IC50 = 0.35 ± 0.8 nM). The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of KTM did not adopt the disulfide fold of α-CTx MII for which it was designed, but instead adopted a flexible ribbon backbone and disulfide connectivity of C2-C16 and C3-C8 with an estimated 12.5% α-helical content. In contrast, α-CTx MII, which has a native fold of C2-C8 and C3-C16, has an estimated 38.1% α-helical secondary structure. KTM is the first reported instance of a Framework I (CC-C-C) α-CTx with ribbon connectivity to display sub-nanomolar inhibitory potency of rα3ß2-nAChR subtypes.

NaV1.4 DI-S4 periodic paralysis mutation R222W enhances inactivation and promotes leak current to attenuate action potentials and depolarize muscle fibers.

Hypokalemic periodic paralysis is a skeletal muscle disease characterized by episodic weakness associated with low serum potassium. We compared clinical and biophysical effects of R222W, the first hNaV1.4 domain I mutation linked to this disease. R222W patients exhibited a higher density of fibers with depolarized resting membrane potentials and produced action potentials that were attenuated compared to controls. Functional characterization of the R222W mutation in heterologous expression included the inactivation deficient IFM/QQQ background to isolate activation. R222W decreased sodium current and slowed activation without affecting probability. Consistent with the phenotype of muscle weakness, R222W shifted fast inactivation to hyperpolarized potentials, promoted more rapid entry, and slowed recovery. R222W increased the extent of slow inactivation and slowed its recovery. A two-compartment skeletal muscle fiber model revealed that defects in fast inactivation sufficiently explain action potential attenuation in patients. Molecular dynamics simulations showed that R222W disrupted electrostatic interactions within the gating pore, supporting the observation that R222W promotes omega current at hyperpolarized potentials. Sodium channel inactivation defects produced by R222W are the primary driver of skeletal muscle fiber action potential attenuation, while hyperpolarization-induced omega current produced by that mutation promotes muscle fiber depolarization.

Kir2.2 p.Thr140Met: a genetic susceptibility to sporadic periodic paralysis.

INTRODUCTION: Periodic paralyses (PP) are recurrent episodes of flaccid limb muscle weakness. Next to autosomal dominant forms, sporadic PP (SPP) cases are known but their genetics are unclear. METHODS: In a patient with hypokalemic SPP, we performed exome sequencing to identify a candidate gene. We sequenced this gene in 263 unrelated PP patients without any known causative mutations. Then we performed functional analysis of all variants found and molecular modelling for interpretation. RESULTS: Exome sequencing in the proband yielded three heterozygous variants predicted to be linked to disease. These encoded p.Thr140Met in the Kir2.2 potassium channel, p.Asp229Asn in protein kinase C theta, and p.Thr15943Ile in titin. Since all hitherto known causative PP genes code for ion channels, we studied the Kir2.2-encoding gene, KCNJ12, for involvement in PP pathogenesis. KCNJ12 screening in 263 PP patients revealed three further variants, each in a single individual and coding for p.Gly419Ser, p.Cys75Tyr, and p.Ile283Val. All four Kir2.2 variants were functionally expressed. Only p.Thr140Met displayed relevant functional alterations, i.e. homo-tetrameric channels produced almost no current, and hetero-tetrameric channels suppressed co-expressed wildtype Kir2.1 in a dominant-negative manner. Molecular modelling showed Kir2.2 p.Thr140Met to reduce movement of potassium ions towards binding sites in the hetero-tetramer pore compatible with a reduced maximal current. MD simulations revealed loss of hydrogen bonding with the p.Thr140Met substitution. DISCUSSION: The electrophysiological findings of p.Thr140Met are similar to those found in thyrotoxic PP caused by Kir2.6 mutations. Also, the homologous Thr140 residue is mutated in Kir2.6. This supports the idea that Kir2.2 p.Thr140Met conveys susceptibility to SPP and should be included in genetic screening.

A novel Ile1455Thr variant in the skeletal muscle sodium channel alpha-subunit in a patient with a severe adult-onset proximal myopathy with electrical myotonia and a patient with mild paramyotonia phenotype.

In sodium channelopathies, a severe fixed myopathy caused by a dominant mutation is rare. We describe two unrelated patients with a novel variant, p.Ile1455Thr, with phenotypes of paramyotonia in one case and fixed proximal myopathy with latent myotonia in another. In-vitro whole cell patch-clamp studies show that the mutation slows inactivation and accelerates recovery, in line with other paramyotonia variants with destabilized fast inactivation as pathomechanism. Additionally, p.IleI1455 causes a loss-of-function by reduced membrane insertion, right-shift of activation, and slowed kinetics. Molecular dynamics simulations comparing wild type and mutant Nav1.4 showed that threonine substitution hindered D4S4 mobility in response to membrane depolarization, consistent with effects of the mutation on channel inactivation. The fixed myopathy is likely to be associated to gain-of-function leading to sodium accumulation, regional edema, T-tubular swelling and mitochondrial stress. A possible contribution of the loss-of-function features towards myotonia and myopathy is discussed.

Domain III S4 in closed-state fast inactivation: insights from a periodic paralysis mutation.

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.

The voltage sensor module in sodium channels.

The mechanism by which voltage-gated ion channels respond to changes in membrane polarization during action potential signaling in excitable cells has been the subject of research attention since the original description of voltage-dependent sodium and potassium flux in the squid giant axon. The cloning of ion channel genes and the identification of point mutations associated with channelopathy diseases in muscle and brain has facilitated an electrophysiological approach to the study of ion channels. Experimental approaches to the study of voltage gating have incorporated the use of thiosulfonate reagents to test accessibility, fluorescent probes, and toxins to define domain-specific roles of voltage-sensing S4 segments. Crystallography, structural and homology modeling, and molecular dynamics simulations have added computational approaches to study the relationship of channel structure to function. These approaches have tested models of voltage sensor translocation in response to membrane depolarization and incorporate the role of negative countercharges in the S1 to S3 segments to define our present understanding of the mechanism by which the voltage sensor module dictates gating particle permissiveness in excitable cells.

NaV1.4 mutations cause hypokalaemic periodic paralysis by disrupting IIIS4 movement during recovery.

Hypokalaemic periodic paralysis is typically associated with mutations of voltage sensor residues in calcium or sodium channels of skeletal muscle. To date, causative sodium channel mutations have been studied only for the two outermost arginine residues in S4 voltage sensor segments of domains I to III. These mutations produce depolarization of skeletal muscle fibres in response to reduced extracellular potassium, owing to an inward cation-selective gating pore current activated by hyperpolarization. Here, we describe mutations of the third arginine, R3, in the domain III voltage sensor i.e. an R1135H mutation which was found in two patients in separate families and a novel R1135C mutation identified in a third patient in another family. Muscle fibres from a patient harbouring the R1135H mutation showed increased depolarization tendency at normal and reduced extracellular potassium compatible with the diagnosis. Additionally, amplitude and rise time of action potentials were reduced compared with controls, even for holding potentials at which all NaV1.4 are fully recovered from inactivation. These findings may be because of an outward omega current activated at positive potentials. Expression of R1135H/C in mammalian cells indicates further gating defects that include significantly enhanced entry into inactivation and prolonged recovery that may additionally contribute to action potential inhibition at the physiological resting potential. After S4 immobilization in the outward position, mutant channels produce an inward omega current that most likely depolarizes the resting potential and produces the hypokalaemia-induced weakness. Gating current recordings reveal that mutations at R3 inhibit S4 deactivation before recovery, and molecular dynamics simulations suggest that this defect is caused by disrupted interactions of domain III S2 countercharges with S4 arginines R2 to R4 during repolarization of the membrane. This work reveals a novel mechanism of disrupted S4 translocation for hypokalaemic periodic paralysis mutations at arginine residues located below the gating pore constriction of the voltage sensor module.

Acetylcholine promotes binding of α-conotoxin MII at α3 ß2 nicotinic acetylcholine receptors.

α-Conotoxin MII (α-CTxMII) is a 16-residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2-Cys8 and Cys3-Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand-binding affinity, homology models of the heteropentameric α3ß2-nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3- and ß2-subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3ß2-nAChR for α-CTxMII with ACh bound to the receptor, and this was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs.

Transient compartment-like syndrome and normokalaemic periodic paralysis due to a Ca(v)1.1 mutation.

We studied a two-generation family presenting with conditions that included progressive permanent weakness, myopathic myopathy, exercise-induced contracture before normokalaemic periodic paralysis or, if localized to the tibial anterior muscle group, transient compartment-like syndrome (painful acute oedema with neuronal compression and drop foot). 23Na and 1H magnetic resonance imaging displayed myoplasmic sodium overload, and oedema. We identified a novel familial Ca(v)1.1 calcium channel mutation, R1242G, localized to the third positive charge of the domain IV voltage sensor. Functional expression of R1242G in the muscular dysgenesis mouse cell line GLT revealed a 28% reduced central pore inward current and a -20 mV shift of the steady-state inactivation curve. Both changes may be at least partially explained by an outward omega (gating pore) current at positive potentials. Moreover, this outward omega current of 27.5 nS/nF may cause the reduction of the overshoot by 13 mV and slowing of the upstroke of action potentials by 36% that are associated with muscle hypoexcitability (permanent weakness and myopathic myopathy). In addition to the outward omega current, we identified an inward omega pore current of 95 nS/nF at negative membrane potentials after long depolarizing pulses that shifts the R1242G residue above the omega pore constriction. A simulation reveals that the inward current might depolarize the fibre sufficiently to trigger calcium release in the absence of an action potential and therefore cause an electrically silent depolarization-induced muscle contracture. Additionally, evidence of the inward current can be found in 23Na magnetic resonance imaging-detected sodium accumulation and 1H magnetic resonance imaging-detected oedema. We hypothesize that the episodes are normokalaemic because of depolarization-induced compensatory outward potassium flux through both delayed rectifiers and omega pore. We conclude that the position of the R1242G residue before elicitation of the omega current is decisive for its conductance: if the residue is located below the gating pore as in the resting state then outward currents are observed; if the residue is above the gating pore because of depolarization, as in the inactivated state, then inward currents are observed. This study shows for the first time that functional characterization of omega pore currents is possible using a cultured cell line expressing mutant Ca(v)1.1 channels. Likewise, it is the first calcium channel mutation for complicated normokalaemic periodic paralysis.

S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation.

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1-S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1-S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I-III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

Pathophysiological role of omega pore current in channelopathies.

In voltage-gated cation channels, a recurrent pattern for mutations is the neutralization of positively charged residues in the voltage-sensing S4 transmembrane segments. These mutations cause dominant ion channelopathies affecting many tissues such as brain, heart, and skeletal muscle. Recent studies suggest that the pathogenesis of associated phenotypes is not limited to alterations in the gating of the ion-conducting alpha pore. Instead, aberrant so-called omega currents, facilitated by the movement of mutated S4 segments, also appear to contribute to symptoms. Surprisingly, these omega currents conduct cations with varying ion selectivity and are activated in either a hyperpolarized or depolarized voltage range. This review gives an overview of voltage sensor channelopathies in general and focuses on pathogenesis of skeletal muscle S4 disorders for which current knowledge is most advanced.

Open- and closed-state fast inactivation in sodium channels: differential effects of a site-3 anemone toxin.

The role of sodium channel closed-state fast inactivation in membrane excitability is not well understood. We compared open- and closed-state fast inactivation, and the gating charge immobilized during these transitions, in skeletal muscle channel hNa(V)1.4. A significant fraction of total charge movement and its immobilization occurred in the absence of channel opening. Simulated action potentials in skeletal muscle fibers were attenuated when pre-conditioned by sub-threshold depolarization. Anthopleurin A, a site-3 toxin that inhibits gating charge associated with the movement of DIVS4, was used to assess the role of this voltage sensor in closed-state fast inactivation. Anthopleurin elicited opposing effects on the gating mode, kinetics and charge immobilized during open- versus closed-state fast inactivation. This same toxin produced identical effects on recovery of channel availability and remobilization of gating charge, irrespective of route of entry into fast inactivation. Our findings suggest that depolarization promoting entry into fast inactivation from open versus closed states provides access to the IFMT receptor via different rate-limiting conformational translocations of DIVS4.

A negative charge in transmembrane segment 1 of domain II of the cockroach sodium channel is critical for channel gating and action of pyrethroid insecticides.

Voltage-gated sodium channels are the primary target of pyrethroids, an important class of synthetic insecticides. Pyrethroids bind to a distinct receptor site on sodium channels and prolong the open state by inhibiting channel deactivation and inactivation. Recent studies have begun to reveal sodium channel residues important for pyrethroid binding. However, how pyrethroid binding leads to inhibition of sodium channel deactivation and inactivation remains elusive. In this study, we show that a negatively charged aspartic acid residue at position 802 (D802) located in the extracellular end of transmembrane segment 1 of domain II (IIS1) is critical for both the action of pyrethroids and the voltage dependence of channel activation. Charge-reversing or -neutralizing substitutions (K, G, or A) of D802 shifted the voltage dependence of activation in the depolarizing direction and reduced channel sensitivity to deltamethrin, a pyrethroid insecticide. The charge-reversing mutation D802K also accelerated open-state deactivation, which may have counteracted the inhibition of sodium channel deactivation by deltamethrin. In contrast, the D802G substitution slowed open-state deactivation, suggesting an additional mechanism for neutralizing the action of deltamethrin. Importantly, Schild analysis showed that D802 is not involved in pyrethroid binding. Thus, we have identified a sodium channel residue that is critical for regulating the action of pyrethroids on the sodium channel without affecting the receptor site of pyrethroids.

Differential effects of paramyotonia congenita mutations F1473S and F1705I on sodium channel gating.

We investigated effects of paramyotonia congenita mutations F1473S and F1705I on gating of skeletal muscle Na+ channels. We used on-cell recordings from Xenopus oocytes to compare fast inactivation and deactivation in wild-type and mutant channels. Then, we used gating current recordings to determine how these actions of PC mutants might be reflected in their effects on charge movement and its immobilization. F1473S, but not F1705I, accelerated deactivation from the inactivated state and enhanced the remobilization of gating charge. F1473S and F1705I decreased the completion of closed-state fast inactivation, and decreased charge movement over the voltage range at which channels did not activate. An unexpected result was that F1705I increased the extent of charge immobilization in response to strong depolarization. Our results suggest that the DIV S4-S5 linker mutation F1473S promotes the hyperpolarized position of DIVS4 to accelerate recovery. Inhibition of charge movement by F1473S and F1705I in the absence of channel opening is discussed with respect to their effects on closed-state fast inactivation.

Charge immobilization of skeletal muscle Na+ channels: role of residues in the inactivation linker.

We investigated structural determinants of fast inactivation and deactivation in sodium channels by comparing ionic flux and charge movement in skeletal muscle channels, using mutations of DIII-DIV linker charges. Charge altering and substituting mutations at K-1317, K-1318 depolarized the g(V) curve but hyperpolarized the h(infinity) curve. Charge reversal and substitution at this locus reduced the apparent voltage sensitivity of open- and closed-state fast inactivation. These effects were not observed with charge reversal at E-1314, E-1315. Mutations swapping or neutralizing the negative cluster at 1314, 1315 and the positive cluster at 1317, 1318 indicated that local interactions dictate the coupling of activation to fast inactivation. Gating charge was immobilized before channel entry into fast inactivation in hNa(V)1.4 but to a lesser extent in mutations at K-1317, K-1318. These results suggest that charge is preferentially immobilized in channels inactivating from the open state. Recovery of gating charge proceeded with a single, fast phase in the double mutation K-1317R, K-1318R. This mutation also partially uncoupled recovery from deactivation. Our findings indicate that charged residues near the fast inactivation "particle" allosterically interact with voltage sensors to control aspects of gating in sodium channels.

Central charged residues in DIIIS4 regulate deactivation gating in skeletal muscle sodium channels.

1. Mutations in the S4 segment of domain III in the voltage gated skeletal muscle sodium channel hNa(V)1.4 were constructed to test the roles of each charged residue in deactivation gating. Mutations comprised charge reversals at K1-R6, charge neutralization, and substitution at R4 and R5. 2. Charge-reversing mutations at R4 and R5 produced the greatest alteration of activation parameters compared to hNa(V)1.4. Effects included depolarization of the conductance/voltage (g/V) curve, decreased valence and slowing of kinetics. 3. Reversal of charge at R2 to R4 hyperpolarized, and reversal at R5 or R6 depolarized the h (infinity) curve. Most DIIIS4 mutations slowed inactivation from the open state. R4E slowed closed state fast inactivation and R5E inhibited its completion .4. Deactivation from the open and/or inactivated state was prolonged in mutations reversing charge at R2 to R4 but accelerated by reversal of charge at R5 or R6. Effects were most pronounced at central charges R4 and R5. 5. Charge and structure each contribute to effects of mutations at R4 and R5 on channel gating. Effects of mutations on activation and deactivation at R4 and, to a lesser extent R5, were primarily owing to charge alteration, whereas effects on fast inactivation were charge independent.