Long-chain scorpion toxins with four disulfide bridges exhibit various pharmacological features towards the different voltage-gated sodium channel subtypes. However, the toxin production still remains a huge challenge. Here, we reported the effects of different expression vectors on the pharmacological properties of a novel toxin BmαTX47 from the scorpion Buthus martensii Karsch. The recombinant BmαTX47 was obtained using the expression vector pET-14b and pET-28a, respectively. Pharmacological experiments showed that the recombinant BmαTX47 was a new α-scorpion toxin which could inhibit the fast inactivation of rNav1.2, mNav1.4 and hNav1.5 channels. Importantly, the different expression vectors were found to strongly affect BmαTX47 pharmacological activities while toxins were obtained by the same expression and purification procedures. When 10 µM recombinant BmαTX47 from the pET-28a vector was applied, the values of I5ms/Ipeak for rNav1.2, mNav1.4 and hNav1.5 channels were 44.12% ± 3.17%, 25.40% ± 4.89% and 65.34% ± 3.86%, respectively, which were better than those values of 11.33% ± 1.46%, 15.96% ± 1.87% and 5.24% ± 2.38% for rNav1.2, mNav1.4 and hNav1.5 channels delayed by 10 µM recombinant BmαTX47 from the pET-14b vector. The dose-response experiments further indicated the EC50 values of recombinant BmαTX47 from the pET-28a vector were 7262.9 ± 755.9 nM for rNav1.2 channel and 1005.8 ± 118.6 nM for hNav1.5 channel, respectively. Together, these findings highlighted the important role of expression vectors in scorpion toxin pharmacological properties, which would accelerate the understanding of the structure-function relationships of scorpion toxins and promote the potential application of toxins in the near future.
Buthus martensii Karsch; BmαTX47; recombinant expression; sodium channels; pET-28a vector; pET-14b vector
BmK IT2 is regarded as a receptor site-4 modulator of sodium channels with depressant insect toxicity. It also displays anti-nociceptive and anti-convulsant activities in rat models. In this study, the potency and efficacy of BmK IT2 were for the first time assessed and compared among four sodium channel isoforms expressed in Xenopus oocytes. Combined with molecular approach, the receptor site of BmK IT2 was further localized.
2 µM BmK IT2 strongly shifted the activation of DmNav1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNav1.2, rNav1.3 and mNav1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu896, Leu899, Gly904 in extracellular loop Domain II S3–S4 of DmNav1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNav1 could be conferred by Domain III. Analysis of subsequent DmNav1 mutants highlighted the residues in Domain III pore loop, esp. Ile1529 was critical for recognition and binding of BmK IT2.
In this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNav1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Nav1.2, Nav1.3 and Nav1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.
β subunits of mammalian sodium channels play important roles in modulating the expression and gating of mammalian sodium channels. However, there are no orthologs of β subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there are four TipE-homologous genes (TEH1-4) in D. melanogaster and three to four orthologs in other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various insect sodium channels expressed in Xenopus oocytes. However, limited information is available on how these proteins modulate the gating of sodium channels, particularly sodium channel variants generated by alternative splicing and RNA editing. In this study, we compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel splice variants, DmNav9-1, DmNav22, and DmNav26, in Xenopus oocytes. Both TipE and TEH1 enhanced the amplitude of sodium current and accelerated current decay of all three sodium channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage-dependence of activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast inactivation of DmNav26. Further analysis of the gating kinetics of DmNav9-1 revealed that TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating sodium channel function and neuronal excitability in vivo.
Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing α-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na+ channel isoforms.
C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Nav1.7, a Na+ channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Nav1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Nav1.2, Nav1.3 and Nav1.4. The effects of CvIV4 are similar to Old World α-toxins that target Nav1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Nav1.7 channels (D1586A and E1589Q, DIV S3–S4 linker) reduced but did not abolish the effects of CvIV4.
This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na+ channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World α-toxins that target Nav1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution.
Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation “temperature-induced-paralysis locus E.” The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na+ currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3′UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an intron retention process in the transcription of the neuronal TEH1-like ancillary subunits of P. americana.
Indoxacarb (DPX-JW062) was recently developed as a new oxadiazine insecticide with high insecticidal activity and low mammalian toxicity. Previous studies showed that indoxacarb and its bioactive metabolite, N-decarbomethoxyllated JW062 (DCJW), block insect sodium channels in nerve preparations and isolated neurons. However, the molecular mechanism of indoxacarb/DCJW action on insect sodium channels is not well understood. In this study, we identified two cockroach sodium channel variants, BgNav1-1 and BgNav1-4, which differ in voltage dependence of fast and slow inactivation, and channel sensitivity to DCJW. The voltage dependence of fast inactivation and slow inactivation of BgNav1-4 were shifted in the hyperpolarizing direction compared with those of BgNav1-1 channels. At the holding potential of −90 mV, 20 μM of DCJW reduced the peak current of BgNav1-4 by about 40%, but had no effect on BgNav1-1. However, at the holding potential of −60 mV, DCJW also reduced the peak currents of BgNav1-1 by about 50%. Furthermore, DCJW delayed the recovery from slow inactivation of both variants. Substitution of E1689 in segment 4 of domain four (IVS4) of BgNav1-4 with a K, which is present in BgNav1-1, was sufficient to shift the voltage dependence of fast and slow inactivation of BgNav1-4 channels to the more depolarizing membrane potential close to that of BgNav1-1 channels. The E1689K change also eliminated the DCJW inhibition of BgNav1-4 at the hyperpolarizing holding potentials. These results show that the E1689K change is responsible for the difference in channel gating and sensitivity to DCJW between BgNav1-4 and BgNav1-1. Our results support the notion that DCJW preferably acts on the inactivated state of the sodium channel and demonstrate that K1689E is a major molecular determinant of the voltage-dependent inactivation and state-dependent action of DCJW.
Insect sodium channel; Insecticide; Indoxacarb; DCJW; Xenopus oocyte
Voltage-activated sodium (Nav) channels are crucial for the generation and propagation of nerve impulses, and as such are amongst the most widely targeted ion channels by toxins and drugs. The four voltage sensors in Nav channels have distinct amino acid sequences, raising fundamental questions about their relative contributions to the function and pharmacology of the channel. Here we use four-fold symmetric voltage-activated potassium (Kv) channels as reporters to examine the contributions of individual Nav channel S3b-S4 paddle motifs to the kinetics of voltage sensor activation and to forming toxin receptors. Our results uncover binding sites for toxins from tarantula and scorpion venom on each of the four paddle motifs in Nav channels and reveal how paddle-specific interactions can be used to reshape Nav channel activity. One paddle motif is unique in that it slows voltage sensor activation and toxins selectively targeting this motif impede Nav channel inactivation. This reporter approach and the principles that emerge will be useful in developing new drugs for treating pain and Nav channelopathies.
Sensory neurons of the dorsal root ganglia (DRG) express multiple voltage-gated sodium (Na) channels that substantially differ in gating kinetics and pharmacology. Small-diameter (<25 µm) neurons isolated from the rat DRG express a combination of fast tetrodotoxin-sensitive (TTX-S) and slow TTX-resistant (TTX-R) Na currents while large-diameter neurons (>30 µm) predominately express fast TTX-S Na current. Na channel expression was further investigated using single-cell RT-PCR to measure the transcripts present in individually harvested DRG neurons. Consistent with cellular electrophysiology, the small neurons expressed transcripts encoding for both TTX-S (Nav1.1, Nav1.2, Nav1.6, Nav1.7) and TTX-R (Nav1.8, Nav1.9) Na channels. Nav1.7, Nav1.8 and Nav1.9 were the predominant Na channels expressed in the small neurons. The large neurons highly expressed TTX-S isoforms (Nav1.1, Nav1.6, Nav1.7) while TTX-R channels were present at comparatively low levels. A unique subpopulation of the large neurons was identified that expressed TTX-R Na current and high levels of Nav1.8 transcript. DRG neurons also displayed substantial differences in the expression of neurofilaments (NF200, peripherin) and Necl-1, a neuronal adhesion molecule involved in myelination. The preferential expression of NF200 and Necl-1 suggests that large-diameter neurons give rise to thick myelinated axons. Small-diameter neurons expressed peripherin, but reduced levels of NF200 and Necl-1, a pattern more consistent with thin unmyelinated axons. Single-cell analysis of Na channel transcripts indicates that TTX-S and TTX-R Na channels are differentially expressed in large myelinated (Nav1.1, Nav1.6, Nav1.7) and small unmyelinated (Nav1.7, Nav1.8, Nav1.9) sensory neurons.
Sodium channel; dorsal root ganglia; single-cell RT-PCR; Necl-1; NF200; peripherin
Voltage-gated sodium channels (NaV) are fundamental components of the nervous system. Their dysfunction is implicated in a number of neurological disorders, such as chronic pain, making them potential targets for the treatment of such disorders. The prominence of the NaV channels in the nervous system has been exploited by venomous animals for preying purposes, which have developed toxins that can block the NaV channels, thereby disabling their function. Because of their potency, such toxins could provide drug leads for the treatment of neurological disorders associated with NaV channels. However, most toxins lack selectivity for a given target NaV channel, and improving their selectivity profile among the NaV1 isoforms is essential for their development as drug leads. Computational methods will be very useful in the solution of such design problems, provided accurate models of the protein-ligand complex can be constructed. Using docking and molecular dynamics simulations, we have recently constructed a model for the NaV1.4-µ-conotoxin-GIIIA complex and validated it with the ample mutational data available for this complex. Here, we use the validated NaV1.4 model in a systematic study of binding other µ-conotoxins (PIIIA, KIIIA and BuIIIB) to NaV1.4. The binding mode obtained for each complex is shown to be consistent with the available mutation data and binding constants. We compare the binding modes of PIIIA, KIIIA and BuIIIB to that of GIIIA and point out the similarities and differences among them. The detailed information about NaV1.4-µ-conotoxin interactions provided here will be useful in the design of new NaV channel blocking peptides.
sodium channels; conotoxins; homology modeling; docking; molecular dynamics; potential of mean force; binding free energy
Several subtypes of voltage-gated Na+ (NaV) channels are important targets for pain management. μ-Conotoxins isolated from venoms of cone snails are potent and specific blockers of different NaV channel isoforms. The inhibitory effect of μ-conotoxins on NaV channels has been examined extensively, but the mechanism of toxin specificity has not been understood in detail. Here the known structure of μ-conotoxin PIIIA and a model of the skeletal muscle channel NaV1.4 are used to elucidate elements that contribute to the structural basis of μ-conotoxin binding and specificity. The model of NaV1.4 is constructed based on the crystal structure of the bacterial NaV channel, NaVAb. Six different binding modes, in which the side chain of each of the basic residues carried by the toxin protrudes into the selectivity filter of NaV1.4, are examined in atomic detail using molecular dynamics simulations with explicit solvent. The dissociation constants (Kd) computed for two selected binding modes in which Lys9 or Arg14 from the toxin protrudes into the filter of the channel are within 2 fold; both values in close proximity to those determined from dose response data for the block of NaV currents. To explore the mechanism of PIIIA specificity, a double mutant of NaV1.4 mimicking NaV channels resistant to μ-conotoxins and tetrodotoxin is constructed and the binding of PIIIA to this mutant channel examined. The double mutation causes the affinity of PIIIA to reduce by two orders of magnitude.
In rats expression of the Nav1.7 voltage-gated sodium channel isoform is restricted to the peripheral nervous system and is abundant in the sensory neurons of the dorsal root ganglion. We expressed the rat Nav1.7 sodium channel α subunit together with the rat auxiliary β1 and β2 subunits in Xenopus laevis oocytes and assessed the effects of the pyrethroid insecticide tefluthrin on the expressed currents using the two-electrode voltage clamp method. Tefluthrin at 100 µM modified of Nav1.7 channels to prolong inactivation of the peak current during a depolarizing pulse, resulting in a marked "late current" at the end of a 40-ms depolarization, and induced a sodium tail current following repolarization. Tefluthrin modification was enhanced up to two-fold by the application of a train of up to 100 5-ms depolarizing prepulses. These effects of tefluthrin on Nav1.7 channels were qualitatively similar to its effects on rat Nav1.2, Nav1.3 and Nav1.6 channels assayed previously under identical conditions. However, Nav1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Nav1.3 and Nav1.6 channels. It is likely that Nav1.7 channels contribute significantly to the tetrodotoxin-sensitive, pyrethroid-resistant current found in cultured dorsal root ganglion neurons. We aligned the complete amino acid sequences of four pyrethroid-sensitive isoforms (house fly Vssc1; rat Nav1.3, Nav1.6 and Nav1.8) and two pyrethroid-resistant isoforms (rat Nav1.2 and Nav1.7) and found only a single site, located in transmembrane segment 6 of homology domain I, at which the amino acid sequence was conserved among all four sensitive isoform sequences but differed in the two resistant isoform sequences. This position, corresponding to Val410 of the house fly Vssc1 sequence, also aligns with sites of multiple amino acid substitutions identified in the sodium channel sequences of pyrethroid-resistant insect populations. These results implicate this single amino acid polymorphism in transmembrane segment 6 of sodium channel homology domain I as a determinant of the differential pyrethroid sensitivity of rat sodium channel isoforms.
voltage-gated sodium channel; Nav1.7 isoform; pyrethroid; tefluthrin; peripheral nervous system; dorsal root ganglion
Voltage-gated sodium channels (VGSCs; NaV1.1–NaV1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as β-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC50 values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on NaV1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50 value of 5.12 nM, compared with IC50 values of 61.7–2700 nM for other heterologously expressed NaV1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on NaV1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.
spider toxin; voltage gated sodium channels; JZTX-V; NaV1.4
Intracellular Ca2+ ([Ca2+]i) can trigger dual-mode regulation of the voltage gated cardiac sodium channel (NaV1.5). The channel components of the Ca2+ regulatory system are the calmodulin (CaM)-binding IQ motif and the Ca2+ sensing EF hand–like (EFL) motif in the carboxyl terminus of the channel. Mutations in either motif have been associated with arrhythmogenic changes in expressed NaV1.5 currents. Increases in [Ca2+]i shift the steady-state inactivation of NaV1.5 in the depolarizing direction and slow entry into inactivated states. Mutation of the EFL (NaV1.54X) shifts inactivation in the hyperpolarizing direction compared with the wild-type channel and eliminates the Ca2+ sensitivity of inactivation gating. Modulation of the steady-state availability of NaV1.5 by [Ca2+]i is more pronounced after the truncation of the carboxyl terminus proximal to the IQ motif (NaV1.5Δ1885), which retains the EFL. Mutating the EFL (NaV1.54X) unmasks CaM-mediated regulation of the kinetics and voltage dependence of inactivation. This latent CaM modulation of inactivation is eliminated by mutation of the IQ motif (NaV1.54X-IQ/AA). The LQT3 EFL mutant channel NaV1.5D1790G exhibits Ca2+ insensitivity and unmasking of CaM regulation of inactivation gating. The enhanced effect of CaM on NaV1.54X gating is associated with significantly greater fluorescence resonance energy transfer between enhanced cyan fluorescent protein–CaM and NaV1.54X channels than is observed with wild-type NaV1.5. Unlike other isoforms of the Na channel, the IQ-CaM interaction in the carboxyl terminus of NaV1.5 is latent under physiological conditions but may become manifest in the presence of disease causing mutations in the CT of NaV1.5 (particularly in the EFL), contributing to the production of potentially lethal ventricular arrhythmias.
voltage-gated sodium channel; EF hand motif; IQ motif; calmodulin; FRET
NaV channels play a crucial role in neuronal and muscle excitability. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of NaV1.2, NaV1.4, and NaV1.5, expressed in cultured mammalian cells. Low pH produced different effects on different channel subtypes. Whereas NaV1.4 exhibited very low sensitivity to acidosis, primarily limited to partial block of macroscopic currents, the effects of low pH on gating in NaV1.2 and NaV1.5 were profound. In NaV1.2 low pH reduced apparent valence of steady-state fast inactivation, shifted the τ(V) to depolarizing potentials and decreased channels availability during onset to slow and use-dependent inactivation (UDI). In contrast, low pH delayed open-state inactivation in NaV1.5, right-shifted the voltage-dependence of window current, and increased channel availability during onset to slow and UDI. These results suggest that protons affect channel availability in an isoform-specific manner. A computer model incorporating these results demonstrates their effects on membrane excitability.
gating; activation; fast inactivation; slow inactivation; patch-clamp; sodium channels
In excitable cells, voltage-gated sodium (NaV) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance1,2. Inactivation is a hallmark of NaV channel function and is critical for control of membrane excitability3, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type NavAb channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of NavAb S6-cysteine mutants4, the pore-lining S6 helices and the intracellular activation gate have undergone significant rearrangements in which one pair of S6 segments has collapsed toward the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type NavAb models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains also shift around the perimeter of the pore module in NavAb, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in NaV channel gating and inactivation. These potential inactivated-state structures provide new insights into NaV channel gating and novel avenues to drug development and therapy for a range of debilitating NaV channelopathies.
Antillatoxin (ATX) is a structurally unique lipopeptide produced by the marine cyanobacterium Lyngbya majuscula. ATX activates voltage-gated sodium channel α-subunits at an undefined recognition site and stimulates sodium influx in neurons. However, the pharmacological properties and selectivity of ATX on the sodium channel α-subunits were not fully characterized.
In this study, we characterized the pharmacological properties and selectivity of ATX in cells heterologously expressing rNav1.2, rNav1.4 or rNav1.5 α-subunits by using the Na+ selective fluorescent dye, sodium-binding benzofuran isophthalate. ATX produced sodium influx in cells expressing each sodium channel α-subunit, whereas two other sodium channel activators, veratridine and brevetoxin-2, were without effect. The ATX potency at rNav1.2, rNav1.4 and rNav1.5 did not differ significantly. Similarly, there were no significant differences in the efficacy for ATX-induced sodium influx between rNav1.2, rNav1.4 and rNav1.5 α-subunits. ATX also produced robust Ca2+ influx relative to other sodium channel activators in the calcium-permeable DEAA mutant of rNav1.4 α-subunit. Finally, we demonstrated that the 8-demethyl-8,9-dihydro-antillatoxin analog was less efficacious and less potent in stimulating sodium influx.
ATX displayed a unique efficacy with respect to stimulation of sodium influx in cells expressing rNav1.2, rNav1.4 and rNav1.5 α-subunits. The efficacy of ATX was distinctive inasmuch as it was not shared by activators of neurotoxin sites 2 and 5 on VGSC α-subunits. Given the unique pharmacological properties of ATX interaction with sodium channel α-subunits, decoding the molecular determinants and mechanism of action of antillatoxin may provide further insight into sodium channel gating mechanisms.
The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na+ channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na+ current (INa). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in INa. Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4 min), there is little effect on INa. However, with longer exposures (4–8 min), there is a large decrease in INa, but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) — MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na+ channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.
hNav1.5; hNav1.5-C373Y; hNav1.4; Methanethiosulfonate (MTSEA); Slow inactivation; Fast inactivation
Mechanical, ischemic, and inflammatory injuries to voltage-gated sodium channel (Nav)-rich membranes of axon initial segments and nodes of Ranvier render Nav channels dangerously leaky. By what means? The behavior of recombinant Nav1.6 (Wang et al., 2009) leads us to postulate that, in neuropathologic conditions, structural degradation of axolemmal bilayer fosters chronically left-shifted Nav channel operation, resulting in ENa rundown. This “sick excitable cell Nav-leak” would encompass left-shifted fast- and slow-mode based persistent INa (i.e., Iwindow and slow-inactivating INa). Bilayer-damage-induced electrophysiological dysfunctions of native-Nav channels, and effects on inhibitors on those channels, should, we suggest, be studied in myelinated axons, exploiting INa(V,t) hysteresis data from sawtooth ramp clamp. We hypothesize that (like dihydropyridines for Ca channels), protective lipophilic Nav antagonists would partition more avidly into disorderly bilayers than into the well-packed bilayers characteristic of undamaged, healthy plasma membrane. Whereas inhibitors using aqueous routes would access all Navs equally, differential partitioning into “sick bilayer” would co-localize lipophilic antagonists with “sick-Nav channels,” allowing for more specific targeting of impaired cells. Molecular fine-tuning of Nav antagonists to favor more avid partitioning into damaged than into intact bilayers could reduce side effects. In potentially salvageable neurons of traumatic and/or ischemic penumbras, in inflammatory neuropathies, in muscular dystrophy, in myocytes of cardiac infarct borders, Nav-leak driven excitotoxicity overwhelms cellular repair mechanisms. Precision-tuning of a lipophilic Nav antagonist for greatest efficacy in mildly damaged membranes could render it suitable for the prolonged continuous administration needed to allow for the remodeling of the excitable membranes, and thus functional recovery.
traumatic brain injury; spinal; riluzole; ranolazine; simulation; modeling
A common genetic variant (rs3812718) in a splice donor consensus sequence within the neuronal sodium channel gene SCN1A (encoding NaV1.1) modulates the proportion of transcripts incorporating either the canonical (5A) or alternative (5N) exon 5. A pharmacogenetic association has been reported whereby increased expression of exon 5N containing NaV1.1 transcripts correlated with lower required doses of phenytoin in epileptics. We tested the hypothesis that SCN1A alternative splicing affects the pharmacology of NaV1.1 channels.
To directly examine biophysical and pharmacological differences between the exon 5 splice variants, we performed whole-cell patch clamp recording of tsA201 cells transiently co-expressing either NaV1.1-5A or NaV1.1-5N with the β1 and β2 accessory subunits. We examined tonic inhibition and use-dependent inhibition of NaV1.1 splice isoforms by phenytoin, carbamazepine, and lamotrigine. We also examined the effects of phenytoin and lamotrigine on channel biophysical properties and determined concentration-response relationships for both splice variants.
We observed no significant differences in voltage-dependence of activation, steady-state inactivation, and recovery from inactivation between splice variants. However, NaV1.1-5N channels exhibited enhanced tonic block by phenytoin and lamotrigine compared to NaV1.1-5A. Additionally, NaV1.1-5N exhibited enhanced use-dependent block by phenytoin and lamotrigine across a range of stimulation frequencies and concentrations. Phenytoin and lamotrigine induced shifts in steady-state inactivation and recovery from fast inactivation for both splice isoforms. No splice isoform differences were observed for channel inhibition by carbamazepine.
These results suggest NaV1.1 channels containing exon 5N are more sensitive to the commonly used antiepileptic drugs phenytoin and lamotrigine.
antiepileptic drugs; ion channel gene defects; alternative splicing
Voltage-gated sodium channels (Nav) mediate neuronal action potentials. Tetrodotoxin inhibits all Nav isoforms, but Nav1.8 and Nav1.9 are relatively tetrodotoxin-resistant (TTX-r) compared to other isoforms. Nav1.8 is highly expressed in dorsal root ganglion neurons and is functionally linked to nociception, but the sensitivity of TTX-r isoforms to inhaled anesthetics is unclear.
The sensitivities of heterologously expressed rat TTX-r Nav1.8 and endogenous tetrodotoxin-sensitive (TTX-s) Nav to the prototypic inhaled anesthetic isoflurane were tested in mammalian ND7/23 cells using patch-clamp electrophysiology.
From a holding potential of −70 mV, isoflurane (0.53±0.06 mM, ~1.8 MAC at 24°C) reduced normalized peak Na+ current (INa) of Nav1.8 to 0.55±0.03 and of endogenous TTX-s Nav to 0.56±0.06. Isoflurane minimally inhibited INa from a holding potential of −140 mV. Isoflurane did not affect voltage-dependence of activation, but significantly shifted voltage-dependence of steady-state inactivation by −6 mV for Nav1.8 and by −7 mV for TTX-s Nav. IC50 values for inhibition of peak INa were 0.67±0.06 mM for Nav1.8 and 0.66±0.09 mM for TTX-s Nav; significant inhibition occurred at clinically relevant concentrations as low as 0.58 MAC. Isoflurane produced use-dependent block of Nav1.8; at a stimulation frequency of 10 Hz, 0.56±0.08 mM isoflurane reduced INa to 0.64±0.01 vs. 0.78±0.01 for control.
Isoflurane inhibited the tetrodotoxin-resistant isoform Nav1.8 with potency comparable to that for endogenous tetrodotoxin-sensitive Nav isoforms, indicating that sensitivity to inhaled anesthetics is conserved across diverse Nav family members. Block of Nav1.8 in dorsal root ganglion neurons could contribute to the effects of inhaled anesthetics on peripheral nociceptive mechanisms.
Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in human embryonic kidney cells was studied by applying the Met-preferring oxidant chloramine-T (ChT) or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of conserved Met residues in the intracellular linkers connecting the membrane-spanning segments of the channel (M1469L and M1470L) also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues (442, 1139, 1154, 1316) were without any noticeable effect. The results of mutagenesis of results, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7) and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met target residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.
Sodium channel; Na+ channel; Inactivation; Methionine oxidation; Chloramine-T; Lucifer Yellow; Patch clamp
Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNaV1.4F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNaV1.4F1705I are incompletely understood. The location of the hNaV1.4F1705I in the CT prompted us to examine the role of Ca2+ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human NaV1.4 channel regulation by Ca2+ and CaM. hNaV1.4F1705I inactivation gating is Ca2+-sensitive compared to wild type hNaV1.4 which is Ca2+ insensitive and the mutant channel exhibits a depolarizing shift of the V1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNaV1.4 channel background (rNaV1.4F1698I) eliminates Ca2+ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca2+ sensitivity of gating between wild type and mutant human and rat NaV1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca2+/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows INa decay in a Ca2+-sensitive fashion. The combination of the altered voltage dependence and kinetics of INa decay contribute to the myotonic phenotype and may involve the Ca2+-sensing apparatus in the CT of NaV1.4.
We compared the clinical and physiological consequences of the novel mutation R878C in a highly conserved pore residue in domain II (S5-S6) of human, hNav1.5, cardiac Na+ channels.
Full clinical evaluation of pedigree members through three generations of a Chinese family combined with SCN5A sequencing from genomic DNA was compared with patch and voltage-clamp results from two independent expression systems.
The four mutation carriers showed bradycardia, and slowed sino-atrial, atrioventricular and intraventricular conduction. Two also showed sick sinus syndrome; two had ST elevation in leads V1 and V2. Unlike WT-hNav1.5, whole-cell patch-clamped HEK293 cells expressing R878C-hNav1.5 showed no detectable Na+ currents (iNa), even with substitution of a similarly charged lysine residue. Voltage-clamped Xenopus oocytes injected with either 0.04 or 1.5 μg μL−1 R878C-hNav1.5 cRNA similarly showed no iNa, yet WT-hNav1.5 cRNA diluted to 0.0004–0.0008 ng μL−1resulted in expression of detectable iNa. iNa was simply determined by the amount of injected WT-hNav1.5: doubling the dose of WT-hNav1.5 cRNA doubled iNa. iNa amplitudes and activation and inactivation characteristics were similar irrespective of whether WT-hNav1.5 cRNA was given alone or combined with equal doses of R878C-hNav1.5 cRNA therefore excluding dominant negative phenotypic effects. Na+ channel function in HEK293 cells transfected with R878C-hNav1.5 was not restored by exposure to mexiletine (200 μm) and lidocaine (100 μm). Fluorescence confocal microscopy using E3-Nav1.5 antibody demonstrated persistent membrane expression of both WT and R878C-hNav1.5. Modelling studies confirmed that such iNa reductions reproduced the SSS phenotype.
Clinical consequences of the novel R878C mutation correlate with results of physiological studies.
cardiac Na+ channels; novel mutation; pore-forming region; SCN5A; sick sinus syndrome
The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4) and S5-S6 in Domain 1 (D1) and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively) move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS). The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH3)3+ trimethylammonium, MTSET) and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS) were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx), but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction) providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage-sensor module.
A new class of sodium channel blocker insecticides (SCBIs), which include indoxacarb, its active metabolite, DCJW, and metaflumizone, preferably block inactivated states of both insect and mammalian sodium channels in a manner similar to that by which local anesthetic (LA) drugs block mammalian sodium channels. A recent study showed that two residues in the cockroach sodium channel, F1817 and Y1824, corresponding to two key LA-interacting residues identified in mammalian sodium channels are not important for the action of SCBIs on insect sodium channels, suggesting unique interactions of SCBIs with insect sodium channels. However, the mechanism of action of LAs on insect sodium channels has not been investigated. In this study, we examined the effects of lidocaine on a cockroach sodium channel variant, BgNav1-1a, and determined whether F1817 and Y1824 are also critical for the action of LAs on insect sodium channels. Lidocaine blocked BgNav1-1a channels in the resting state with potency similar to that observed in mammalian sodium channels. Lidocaine also stabilized both fast-inactivated and slow-inactivated states of BgNav1-1a channels, and caused a limited degree of use- and frequency-dependent block, major characteristics of LA action on mammalian sodium channels. Alanine substitutions of F1817 and Y1824 reduced the sensitivity of the BgNav1-1a channel to the use-dependent block by lidocaine, but not to tonic blocking and inactivation stabilizing effects of lidocaine. Thus, similar to those on mammalian sodium channels, F1817 and Y1824 are important for the action of lidocaine on cockroach sodium channels. Our results suggest that the receptor sites for lidocaine and SCBIs are different on insect sodium channels.
lidocaine; LA receptor site; insect sodium channel