Structural basis for severe pain caused by mutations in the voltage sensors of sodium channel NaV1.7.

Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.

A severe case of primary erythromelalgia presenting as small fiber neuropathy with a novel SCN9A mutation.

Primary erythromelalgia (PEM) is a rare condition characterized by severe burning pain, erythema, and increased temperature in the extremeties. Mutations in the Nav1.7 sodium channel encoded by the SCN9A are responsible for PEM. The pathophysiology of PEM is unclear, but the involvement of neurogenic and vasogenic mechanisms has been suggested. Here we report a case of severe PEM in a 9-year-old child with a novel SCN9A mutation and examine the distribution of nerve fibers and expression of neuropeptides in the affected skin. Gene mutation analysis revealed a novel mutation p.L951I (c.2851C>A) in the heterozygous form of the SCN9A. An immunofluorescence study showed that intraepidermal nerve fibers were decreased in the affected leg, suggesting small fiber neuropathy. There was no increase in the expression of substance P (SP) or calcitonin gene-related peptide (CGRP) in the lesional skin tissue. These findings suggest SP and CGRP do not play a major role in the pathophysiology of primary erythromelalgia.

Cryo-EM structure of human voltage-gated sodium channel Nav1.6.

Voltage-gated sodium channel Nav1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Nav1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Nav1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Nav1.6 in the presence of auxiliary subunits ß1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the ß1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Nav1.6-specific blockers. Structural mapping of disease-related Nav1.6 mutations provides insights into their pathogenic mechanism.

Unwinding and spiral sliding of S4 and domain rotation of VSD during the electromechanical coupling in Nav1.7.

Voltage-gated sodium (Nav) channel Nav1.7 has been targeted for the development of nonaddictive pain killers. Structures of Nav1.7 in distinct functional states will offer an advanced mechanistic understanding and aid drug discovery. Here we report the cryoelectron microscopy analysis of a human Nav1.7 variant that, with 11 rationally introduced point mutations, has a markedly right-shifted activation voltage curve with V1/2 reaching 69 mV. The voltage-sensing domain in the first repeat (VSDI) in a 2.7-Å resolution structure displays a completely down (deactivated) conformation. Compared to the structure of WT Nav1.7, three gating charge (GC) residues in VSDI are transferred to the cytosolic side through a combination of helix unwinding and spiral sliding of S4I and ∼20° domain rotation. A conserved WNФФD motif on the cytoplasmic end of S3I stabilizes the down conformation of VSDI. One GC residue is transferred in VSDII mainly through helix sliding. Accompanying GC transfer in VSDI and VSDII, rearrangement and contraction of the intracellular gate is achieved through concerted movements of adjacent segments, including S4-5I, S4-5II, S5II, and all S6 segments. Our studies provide important insight into the electromechanical coupling mechanism of the single-chain voltage-gated ion channels and afford molecular interpretations for a number of pain-associated mutations whose pathogenic mechanism cannot be revealed from previously reported Nav structures.

Structural basis for high-voltage activation and subtype-specific inhibition of human Nav1.8.

The dorsal root ganglia-localized voltage-gated sodium (Nav) channel Nav1.8 represents a promising target for developing next-generation analgesics. A prominent characteristic of Nav1.8 is the requirement of more depolarized membrane potential for activation. Here we present the cryogenic electron microscopy structures of human Nav1.8 alone and bound to a selective pore blocker, A-803467, at overall resolutions of 2.7 to 3.2 Å. The first voltage-sensing domain (VSDI) displays three different conformations. Structure-guided mutagenesis identified the extracellular interface between VSDI and the pore domain (PD) to be a determinant for the high-voltage dependence of activation. A-803467 was clearly resolved in the central cavity of the PD, clenching S6IV. Our structure-guided functional characterizations show that two nonligand binding residues, Thr397 on S6I and Gly1406 on S6III, allosterically modulate the channel's sensitivity to A-803467. Comparison of available structures of human Nav channels suggests the extracellular loop region to be a potential site for developing subtype-specific pore-blocking biologics.

Structural basis for modulation of human NaV1.3 by clinical drug and selective antagonist.

Voltage-gated sodium (NaV) channels play fundamental roles in initiating and propagating action potentials. NaV1.3 is involved in numerous physiological processes including neuronal development, hormone secretion and pain perception. Here we report structures of human NaV1.3/ß1/ß2 in complex with clinically-used drug bulleyaconitine A and selective antagonist ICA121431. Bulleyaconitine A is located around domain I-II fenestration, providing the detailed view of the site-2 neurotoxin binding site. It partially blocks ion path and expands the pore-lining helices, elucidating how the bulleyaconitine A reduces peak amplitude but improves channel open probability. In contrast, ICA121431 preferentially binds to activated domain IV voltage-sensor, consequently strengthens the Ile-Phe-Met motif binding to its receptor site, stabilizes the channel in inactivated state, revealing an allosterically inhibitory mechanism of NaV channels. Our results provide structural details of distinct small-molecular modulators binding sites, elucidate molecular mechanisms of their action on NaV channels and pave a way for subtype-selective therapeutic development.

Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties.

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.

3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM.

Single particle cryo-EM excels in determining static structures of protein molecules, but existing 3D reconstruction methods have been ineffective in modelling flexible proteins. We introduce 3D variability analysis (3DVA), an algorithm that fits a linear subspace model of conformational change to cryo-EM data at high resolution. 3DVA enables the resolution and visualization of detailed molecular motions of both large and small proteins, revealing new biological insight from single particle cryo-EM data. Experimental results demonstrate the ability of 3DVA to resolve multiple flexible motions of α-helices in the sub-50 kDa transmembrane domain of a GPCR complex, bending modes of a sodium ion channel, five types of symmetric and symmetry-breaking flexibility in a proteasome, large motions in a spliceosome complex, and discrete conformational states of a ribosome assembly. 3DVA is implemented in the cryoSPARC software package.

Engineering of highly potent and selective HNTX-III mutant against hNav1.7 sodium channel for treatment of pain.

Human voltage-gated sodium channel Nav1.7 (hNav1.7) is involved in the generation and conduction of neuropathic and nociceptive pain signals. Compelling genetic and preclinical studies have validated that hNav1.7 is a therapeutic target for the treatment of pain; however, there is a dearth of currently available compounds capable of targeting hNav1.7 with high potency and specificity. Hainantoxin-III (HNTX-III) is a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. Here, we report the engineering of improved potency and Nav selectivity of hNav1.7 inhibition peptides derived from the HNTX-III scaffold. Alanine scanning mutagenesis showed key residues for HNTX-III interacting with hNav1.7. Site-directed mutagenesis analysis indicated key residues on hNav1.7 interacting with HNTX-III. Molecular docking was conducted to clarify the binding interface between HNTX-III and Nav1.7 and guide the molecular engineering process. Ultimately, we obtained H4 [K0G1-P18K-A21L-V] based on molecular docking of HNTX-III and hNav1.7 with a 30-fold improved potency (IC50 0.007 ± 0.001 µM) and >1000-fold selectivity against Nav1.4 and Nav1.5. H4 also showed robust analgesia in the acute and chronic inflammatory pain model and neuropathic pain model. Thus, our results provide further insight into peptide toxins that may prove useful in guiding the development of inhibitors with improved potency and selectivity for Nav subtypes with robust analgesia.

Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity.

Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain.

Structural Basis for High-Affinity Trapping of the NaV1.7 Channel in Its Resting State by Tarantula Toxin.

Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaVAb with voltage-shifting mutations and the toxin-binding site of human NaV1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.

Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated µ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.

Computational pipeline to probe NaV1.7 gain-of-function variants in neuropathic painful syndromes.

Applications of machine learning and graph theory techniques to neuroscience have witnessed an increased interest in the last decade due to the large data availability and unprecedented technology developments. Their employment to investigate the effect of mutational changes in genes encoding for proteins modulating the membrane of excitable cells, whose biological correlates are assessed at electrophysiological level, could provide useful predictive clues. We apply this concept to the analysis of variants in sodium channel NaV1.7 subunit found in patients with chronic painful syndromes, by the implementation of a dedicated computational pipeline empowering different and complementary techniques including homology modeling, network theory, and machine learning. By testing three templates of different origin and sequence identities, we provide an optimal condition for its use. Our findings reveal the usefulness of our computational pipeline in supporting the selection of candidates for cell electrophysiology assay and with potential clinical applications.

Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins.

The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1-1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.

Application of Pharmacokinetic-Pharmacodynamic Modeling to Inform Translation of In Vitro NaV1.7 Inhibition to In Vivo Pharmacological Response in Non-human Primate.

PURPOSE: This work describes a staged approach to the application of pharmacokinetic-pharmacodynamic (PK-PD) modeling in the voltage-gated sodium ion channel (NaV1.7) inhibitor drug discovery effort to address strategic questions regarding in vitro to in vivo translation of target modulation. METHODS: PK-PD analysis was applied to data from a functional magnetic resonance imaging (fMRI) technique to non-invasively measure treatment mediated inhibition of olfaction signaling in non-human primates (NHPs). Initial exposure-response was evaluated using single time point data pooled across 27 compounds to inform on in vitro to in vivo correlation (IVIVC). More robust effect compartment PK-PD modeling was conducted for a subset of 10 compounds with additional PD and PK data to characterize hysteresis. RESULTS: The pooled compound exposure-response facilitated an early exploration of IVIVC with a limited dataset for each individual compound, and it suggested a 2.4-fold in vitro to in vivo scaling factor for the NaV1.7 target. Accounting for hysteresis with an effect compartment PK-PD model as compounds advanced towards preclinical development provided a more robust determination of in vivo potency values, which resulted in a statistically significant positive IVIVC with a slope of 1.057 ± 0.210, R-squared of 0.7831, and p value of 0.006. Subsequent simulations with the PK-PD model informed the design of anti-nociception efficacy studies in NHPs. CONCLUSIONS: A staged approach to PK-PD modeling and simulation enabled integration of in vitro NaV1.7 potency, plasma protein binding, and pharmacokinetics to describe the exposure-response profile and inform future study design as the NaV1.7 inhibitor effort progressed through drug discovery.

Conservation and divergence in NaChBac and NaV1.7 pharmacology reveals novel drug interaction mechanisms.

Voltage-gated Na+ (NaV) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts, bacterial NaV channels possess a simpler, fourfold symmetric structure and have facilitated studies of the structural basis of channel gating. However, the pharmacology of bacterial NaV remains largely unexplored. Here we systematically screened 39 NaV modulators on a bacterial channel (NaChBac) and characterized a selection of compounds on NaChBac and a mammalian channel (human NaV1.7). We found that while many compounds interact with both channels, they exhibit distinct functional effects. For example, the local anesthetics ambroxol and lidocaine block both NaV1.7 and NaChBac but affect activation and inactivation of the two channels to different extents. The voltage-sensing domain targeting toxin BDS-I increases NaV1.7 but decreases NaChBac peak currents. The pore binding toxins aconitine and veratridine block peak currents of NaV1.7 and shift activation (aconitine) and inactivation (veratridine) respectively. In NaChBac, they block the peak current by binding to the pore residue F224. Nonetheless, aconitine has no effect on activation or inactivation, while veratridine only modulates activation of NaChBac. The conservation and divergence in the pharmacology of bacterial and mammalian NaV channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.

Discovery of Potent, Selective, and State-Dependent NaV1.7 Inhibitors with Robust Oral Efficacy in Pain Models: Structure-Activity Relationship and Optimization of Chroman and Indane Aryl Sulfonamides.

Voltage-gated sodium channel NaV1.7 is a genetically validated target for pain. Identification of NaV1.7 inhibitors with all of the desired properties to develop as an oral therapeutic for pain has been a major challenge. Herein, we report systematic structure-activity relationship (SAR) studies carried out to identify novel sulfonamide derivatives as potent, selective, and state-dependent NaV1.7 inhibitors for pain. Scaffold hopping from benzoxazine to chroman and indane bicyclic system followed by thiazole replacement on sulfonamide led to identification of lead molecules with significant improvement in solubility, selectivity over NaV1.5, and CYP2C9 inhibition. The lead molecules 13, 29, 32, 43, and 51 showed a favorable pharmacokinetics (PK) profile across different species and robust efficacy in veratridine and formalin-induced inflammatory pain models in mice. Compound 51 also showed significant effects on the CCI-induced neuropathic pain model. The profile of 51 indicated that it has the potential for further evaluation as a therapeutic for pain.

Spider venom-derived peptide induces hyperalgesia in Nav1.7 knockout mice by activating Nav1.9 channels.

The sodium channels Nav1.7, Nav1.8 and Nav1.9 are critical for pain perception in peripheral nociceptors. Loss of function of Nav1.7 leads to congenital insensitivity to pain in humans. Here we show that the spider peptide toxin called HpTx1, first identified as an inhibitor of Kv4.2, restores nociception in Nav1.7 knockout (Nav1.7-KO) mice by enhancing the excitability of dorsal root ganglion neurons. HpTx1 inhibits Nav1.7 and activates Nav1.9 but does not affect Nav1.8. This toxin produces pain in wild-type (WT) and Nav1.7-KO mice, and attenuates nociception in Nav1.9-KO mice, but has no effect in Nav1.8-KO mice. These data indicate that HpTx1-induced hypersensitivity is mediated by Nav1.9 activation and offers pharmacological insight into the relationship of the three Nav channels in pain signalling.

Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7.

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV1.7 and with an in vivo efficacy similar to that of native HwTx-IV.

Complementary roles of murine NaV1.7, NaV1.8 and NaV1.9 in acute itch signalling.

Acute pruritus occurs in various disorders. Despite severe repercussions on quality of life treatment options remain limited. Voltage-gated sodium channels (NaV) are indispensable for transformation and propagation of sensory signals implicating them as drug targets. Here, NaV1.7, 1.8 and 1.9 were compared for their contribution to itch by analysing NaV-specific knockout mice. Acute pruritus was induced by a comprehensive panel of pruritogens (C48/80, endothelin, 5-HT, chloroquine, histamine, lysophosphatidic acid, trypsin, SLIGRL, ß-alanine, BAM8-22), and scratching was assessed using a magnet-based recording technology. We report an unexpected stimulus-dependent diversity in NaV channel-mediated itch signalling. NaV1.7-/- showed substantial scratch reduction mainly towards strong pruritogens. NaV1.8-/- impaired histamine and 5-HT-induced scratching while NaV1.9 was involved in itch signalling towards 5-HT, C48/80 and SLIGRL. Furthermore, similar microfluorimetric calcium responses of sensory neurons and expression of itch-related TRP channels suggest no change in sensory transduction but in action potential transformation and conduction. The cumulative sum of scratching over all pruritogens confirmed a leading role of NaV1.7 and indicated an overall contribution of NaV1.9. Beside the proposed general role of NaV1.7 and 1.9 in itch signalling, scrutiny of time courses suggested NaV1.8 to sustain prolonged itching. Therefore, NaV1.7 and 1.9 may represent targets in pruritus therapy.