Role of NaV1.7 in postganglionic sympathetic nerve function in human and guinea-pig arteries.

NaV1.7 plays a crucial role in inducing and conducting action potentials in pain-transducing sensory nociceptor fibres, suggesting that NaV1.7 blockers could be effective non-opioid analgesics. While SCN9A is expressed in both sensory and autonomic neurons, its functional role in the autonomic system remains less established. Our single neuron rt-PCR analysis revealed that 82% of sympathetic neurons isolated from guinea-pig stellate ganglia expressed NaV1.7 mRNA, with NaV1.3 being the only other tetrodotoxin-sensitive channel expressed in approximately 50% of neurons. We investigated the role of NaV1.7 in conducting action potentials in postganglionic sympathetic nerves and in the sympathetic adrenergic contractions of blood vessels using selective NaV1.7 inhibitors. Two highly selective NaV1.7 blockers, GNE8493 and PF 05089771, significantly inhibited postganglionic compound action potentials by approximately 70% (P < 0.01), with residual activity being blocked by the NaV1.3 inhibitor, ICA 121431. Electrical field stimulation (EFS) induced rapid contractions in guinea-pig isolated aorta, pulmonary arteries, and human isolated pulmonary arteries via stimulation of intrinsic nerves, which were inhibited by prazosin or the NaV1 blocker tetrodotoxin. Our results demonstrated that blocking NaV1.7 with GNE8493, PF 05089771, or ST2262 abolished or strongly inhibited sympathetic adrenergic responses in guinea-pigs and human vascular smooth muscle. These findings support the hypothesis that pharmacologically inhibiting NaV1.7 could potentially reduce sympathetic and parasympathetic function in specific vascular beds and airways. KEY POINTS: 82% of sympathetic neurons isolated from the stellate ganglion predominantly express NaV1.7 mRNA. NaV1.7 blockers inhibit action potential conduction in postganglionic sympathetic nerves. NaV1.7 blockade substantially inhibits sympathetic nerve-mediated adrenergic contractions in human and guinea-pig blood vessels. Pharmacologically blocking NaV1.7 profoundly affects sympathetic and parasympathetic responses in addition to sensory fibres, prompting exploration into the broader physiological consequences of NaV1.7 mutations on autonomic nerve activity.

Examination of the contribution of Nav1.7 to axonal propagation in nociceptors.

ABSTRACT: Nav1.7 is a promising drug target for the treatment of pain. However, there is a mismatch between the analgesia produced by Nav1.7 loss-of-function and the peripherally restricted Nav1.7 inhibitors, which may reflect a lack of understanding of the function of Nav1.7 in the transmission of nociceptive information. In the periphery, the role of Nav1.7 in transduction at nociceptive peripheral terminals has been comprehensively examined, but its role in axonal propagation in these neurons is less clearly defined. In this study, we examined the contribution of Nav1.7 to axonal propagation in nociceptors using sodium channel blockers in in vivo electrophysiological and calcium imaging recordings in mice. Using the sodium channel blocker tetrodotoxin (TTX) (1-10 µM) to inhibit Nav1.7 and other tetrodotoxin-sensitive sodium channels along the sciatic nerve, we first showed that around two-thirds of nociceptive L4 dorsal root ganglion neurons innervating the skin, but a lower proportion innervating the muscle (45%), are blocked by TTX. By contrast, nearly all large-sized cutaneous afferents (95%-100%) were blocked by axonal TTX. Many cutaneous nociceptors resistant to TTX were polymodal (57%) and capsaicin sensitive (57%). Next, we applied PF-05198007 (300 nM-1 µM) to the sciatic nerve between stimulating and recording sites to selectively block axonal Nav1.7 channels. One hundred to three hundred nanomolar PF-05198007 blocked propagation in 63% of C-fiber sensory neurons, whereas similar concentrations produced minimal block (5%) in rapidly conducting A-fiber neurons. We conclude that Nav1.7 is essential for axonal propagation in around two-thirds of nociceptive cutaneous C-fiber neurons and a lower proportion (≤45%) of nociceptive neurons innervating muscle.

Allium macrostemon Bunge. exerts analgesic activity by inhibiting NaV1.7 channel.

ETHNOPHARMACOLOGICAL RELEVANCE: Allium macrostemon Bunge. is an edible Chinese herb traditionally used for the treatment of thoracic pain, stenocardia, heart asthma and diarrhea. Although its biological potential has been extensively proven such as antioxidant activity, antiplatelet aggregation, vasodilation and antidepressant-like activity, there are no reports in the literature regarding its pharmacological analgesic activity. AIM OF THE STUDY: The study was carried out to examine the anti-nociceptive activity of the crude extract of A. macrostemon bulbs and interpret its likely molecular target. MATERIALS AND METHODS: The bulbs of A. macrostemon were gathered, dried-up, and extracted with water (AMWD). AMWD was subjected to activity testing, using chemical-induced (acetic acid and formalin test) and heat-induced (hot plate) pain models. To evaluate the likely mechanistic strategy involved in the analgesic effect of AMWD, whole-cell patch clamp recordings were conducted in acutely dissociated dorsal root ganglion (DRG) neurons and human embryonic kidney 293T (HEK293T) cells expressing pain-related receptors. Electrophysiological methods were employed to detect the action potentials of DRG neurons and potential targets of A. macrostemon. RESULTS: AMWD showed significant palliative effect in all heat and chemical induced pain assays. Moreover, AMWD significantly reduces the excitability of dorsal root ganglion neurons by reducing the firing frequency of action potentials. Further analysis revealed that voltage-gated sodium channel Nav1.7 is the potential target of A. macrostemon for its analgesic activity. CONCLUSION: This study has brought new scientific evidence of preclinical efficacy of A. macrostemon as an anti-nociceptive agent. Apparently, these effects are involved with the inhibition of the voltage-sensitive Nav1.7 channel contributing to the reduction of peripheral neuronal excitability. Our present study justifies the folkloric usage of A. macrostemon as a remedy for several pain states. Furthermore, A. macrostemon is a good resource for the development of analgesic drugs targeting Nav1.7 channel.

The insecticide deltamethrin enhances sodium channel slow inactivation of human Nav1.9, Nav1.8 and Nav1.7.

The insecticide deltamethrin of the pyrethroid class mainly targets voltage-gated sodium channels (Navs). Deltamethrin prolongs the opening of Navs by slowing down fast inactivation and deactivation. Pyrethroids are supposedly safe for humans, however, they have also been linked to the gulf-war syndrome, a neuropathic pain condition that can develop following exposure to certain chemicals. Inherited neuropathic pain conditions have been linked to mutations in the Nav subtypes Nav1.7, Nav1.8, and Nav1.9. Here, we examined the effect of deltamethrin on the human isoforms Nav1.7, Nav1.8, and Nav1.9_C4 (chimera containing the C-terminus of rat Nav1.4) heterologously expressed in HEK293T and ND7/23 cells using whole-cell patch-clamp electrophysiology. For all three Nav subtypes, we observed increased persistent and tail currents that are typical for Nav channels modified by deltamethrin. The most surprising finding was an enhanced slow inactivation induced by deltamethrin in all three Nav subtypes. An enhanced slow inactivation is contrary to the prolonged opening caused by pyrethroids and has not been described for deltamethrin or any other pyrethroid before. Furthermore, we found that the fraction of deltamethrin-modified channels increased use-dependently. However, for Nav1.8, the use-dependent potentiation occurred only when the holding potential was increased to -90 mV, a potential at which the tail currents decay more slowly. This indicates that use-dependent modification is due to an accumulation of tail currents. In summary, our findings support a novel mechanism whereby deltamethrin enhances slow inactivation of voltage-gated sodium channels, which may, depending on the cellular resting membrane potential, reduce neuronal excitability and counteract the well-described pyrethroid effects of prolonging channel opening.

Two Novel Peptide Toxins from the Spider Cyriopagopus longipes Inhibit Tetrodotoxin-Sensitive Sodium Channels.

Sodium channels play a critical role in the generation and propagation of action potentials in excitable tissues, such as nerves, cardiac muscle, and skeletal muscle, and are the primary targets of toxins found in animal venoms. Here, two novel peptide toxins (Cl6a and Cl6b) were isolated from the venom of the spider Cyriopagopus longipes and characterized. Cl6a and Cl6b were shown to be inhibitors of tetrodotoxin-sensitive (TTX-S), but not TTX-resistant, sodium channels. Among the TTX-S channels investigated, Cl6a and Cl6b showed the highest degree of inhibition against NaV1.7 (half-maximal inhibitory concentration (IC50) of 11.0 ± 2.5 nM and 18.8 ± 2.4 nM, respectively) in an irreversible manner that does not alter channel activation, inactivation, or repriming kinetics. Moreover, analysis of NaV1.7/NaV1.8 chimeric channels revealed that Cl6b is a site 4 neurotoxin. Site-directed mutagenesis analysis indicated that D816, V817, and E818 observably affected the efficacy of the Cl6b-NaV1.7 interaction, suggesting that these residues might directly affect the interaction of NaV1.7 with Cl6b. Taken together, these two novel peptide toxins act as potent and sustained NaV1.7 blockers and may have potential in the pharmacological study of sodium channels.

Differential effect of lacosamide on Nav1.7 variants from responsive and non-responsive patients with small fibre neuropathy.

Small fibre neuropathy is a common pain disorder, which in many cases fails to respond to treatment with existing medications. Gain-of-function mutations of voltage-gated sodium channel Nav1.7 underlie dorsal root ganglion neuronal hyperexcitability and pain in a subset of patients with small fibre neuropathy. Recent clinical studies have demonstrated that lacosamide, which blocks sodium channels in a use-dependent manner, attenuates pain in some patients with Nav1.7 mutations; however, only a subgroup of these patients responded to the drug. Here, we used voltage-clamp recordings to evaluate the effects of lacosamide on five Nav1.7 variants from patients who were responsive or non-responsive to treatment. We show that, at the clinically achievable concentration of 30 µM, lacosamide acts as a potent sodium channel inhibitor of Nav1.7 variants carried by responsive patients, via a hyperpolarizing shift of voltage-dependence of both fast and slow inactivation and enhancement of use-dependent inhibition. By contrast, the effects of lacosamide on slow inactivation and use-dependence in Nav1.7 variants from non-responsive patients were less robust. Importantly, we found that lacosamide selectively enhances fast inactivation only in variants from responders. Taken together, these findings begin to unravel biophysical underpinnings that contribute to responsiveness to lacosamide in patients with small fibre neuropathy carrying select Nav1.7 variants.

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.

Chemical Synthesis, Proper Folding, Nav Channel Selectivity Profile and Analgesic Properties of the Spider Peptide Phlotoxin 1.

Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.

Effects of a State- and Use-Dependent Nav1.7 Channel Blocker on Ambulatory Blood Pressure: A Randomized, Controlled Crossover Study.

Vixotrigine is a state- and use-dependent Nav1.7 channel blocker being investigated for the treatment of neuropathic pain conditions. This randomized, double-blind, placebo-controlled crossover trial was designed to evaluate changes in blood pressure with the administration of vixotrigine using ambulatory blood pressure monitoring (ABPM). Eligible participants were healthy adults 18 to 65 years of age without evidence of baseline systolic blood pressure (SBP) persistently > 140 mm Hg or diastolic blood pressure (DBP) persistently > 90 mm Hg. Vixotrigine (400 mg [men], 300 mg [women]) or placebo was administered orally twice daily for 36 days. Following a 7-day washout period, participants crossed over to the other treatment. Each dosing period was preceded by 1 inpatient visit and 1 outpatient baseline visit. Two 14-hour inpatient ABPM sessions occurred on days 14 and 35, with a return to the clinic the morning of days 15 and 36 for initiation of outpatient ABPM, which assessed blood pressure and heart rate every 15 minutes. Adverse events were collected throughout the study. The primary end point was the change from baseline in 24-hour mean SBP and DBP on day 36. Sixty participants were enrolled; 10 withdrew from the study owing to adverse events, investigator discretion, or withdrawal of consent. From baseline to day 36, mean changes in average SBP and DBP (vixotrigine treated) were -0.33 and 0.20 mm Hg, respectively. Adverse event rates were comparable for vixotrigine and placebo; the most common adverse events were headache, dizziness, and nausea. Vixotrigine administration is not associated with a clinically important increase in blood pressure.

Engineering Gain-of-Function Analogues of the Spider Venom Peptide HNTX-I, A Potent Blocker of the hNaV1.7 Sodium Channel.

Pain is a medical condition that interferes with normal human life and work and reduces human well-being worldwide. The voltage-gated sodium channel (VGSC) human NaV1.7 (hNaV1.7) is a compelling target that plays a key role in human pain signaling. The 33-residue peptide µ-TRTX-Hhn2b (HNTX-I), a member of NaV-targeting spider toxin (NaSpTx) family 1, has shown negligible activity on mammalian VGSCs, including the hNaV1.7 channel. We engineered analogues of HNTX-I based on sequence conservation in NaSpTx family 1. Substitution of Asn for Ser at position 23 or Asp for His at position 26 conferred potent activity against hNaV1.7. Moreover, multiple site mutations combined together afforded improvements in potency. Ultimately, we generated an analogue E1G⁻N23S⁻D26H⁻L32W with >300-fold improved potency compared with wild-type HNTX-1 on hNaV1.7 (IC50 0.036 ± 0.007 µM). Structural simulation suggested that the charged surface and the hydrophobic surface of the modified peptide are responsible for binding affinity to the hNaV1.7 channel, while variable residues may determine pharmacological specificity. Therefore, this study provides a profile for drug design targeting the hNaV1.7 channel.

GpTx-1 and [Ala5 , Phe6 , Leu26 , Arg28 ]GpTx-1, two peptide NaV 1.7 inhibitors: analgesic and tolerance properties at the spinal level.

BACKGROUND AND PURPOSE: The voltage-gated sodium channel NaV 1.7 is considered a therapeutic target for pain treatment based on human genetic evidence. GpTx-1 and its potent analogue [Ala5 , Phe6 , Leu26 , Arg28 ]GpTx-1 (GpTx-1-71) were recently characterized as NaV 1.7 inhibitors in vitro. Furthermore, the present work was conducted to investigate the analgesic properties of these two peptides in different pain models after spinal administration. EXPERIMENTAL APPROACH: The antinociceptive activities of both GpTx-1 and GpTx-1-71 were investigated in mouse models of acute, visceral, inflammatory and neuropathic pain. Furthermore, the side effects of GpTx-1 and GpTx-1-71 were evaluated in rotarod, antinociceptive tolerance, acute hyperlocomotion and gastrointestinal transit tests. KEY RESULTS: The i.t. administration of both GpTx-1 and GpTx-1-71 dose-dependently produced powerful antinociception in the different pain models. This effect was attenuated by the opioid receptor antagonist naloxone, suggesting the involvement of the opioid system. In this study, repeated administration of these two_peptides produced spinal analgesia without a loss of potency over 8 days in mouse models of acute, inflammatory and neuropathic pain. Moreover, spinal administration of GpTx-1 and GpTx-1-71 did not induce significant effects on motor coordination, evoke acute hyperlocomotion or increase gastrointestinal transit time. CONCLUSIONS AND IMPLICATIONS: Our data indicate that the NaV 1.7 peptide inhibitors GpTx-1 and GpTx-1-71 produce powerful, nontolerance-forming analgesia in preclinical pain models, which might be dependent on the endogenous opioid system. In addition, at the spinal level, the limited side effects imply that these NaV 1.7 peptide inhibitors could be potentially developed as GpTx-1-based drugs for pain relief.

ß1 subunit stabilises sodium channel Nav1.7 against mechanical stress.

KEY POINTS: The voltage-gated sodium channel Nav1.7 is a key player in neuronal excitability and pain signalling. In addition to voltage sensing, the channel is also modulated by mechanical stress. Using whole-cell patch-clamp experiments, we discovered that the sodium channel subunit ß1 is able to prevent the impact of mechanical stress on Nav1.7. An intramolecular disulfide bond of ß1 was identified to be essential for stabilisation of inactivation, but not activation, against mechanical stress using molecular dynamics simulations, homology modelling and site-directed mutagenesis. Our results highlight the role of segment 6 of domain IV in fast inactivation. We present a candidate mechanism for sodium channel stabilisation against mechanical stress, ensuring reliable channel functionality in living systems. ABSTRACT: Voltage-gated sodium channels are key players in neuronal excitability and pain signalling. Precise gating of these channels is crucial as even small functional alterations can lead to pathological phenotypes such as pain or heart failure. Mechanical stress has been shown to affect sodium channel activation and inactivation. This suggests that stabilising components are necessary to ensure precise channel gating in living organisms. Here, we show that mechanical shear stress affects voltage dependence of activation and fast inactivation of the Nav1.7 channel. Co-expression of the ß1 subunit, however, protects both gating modes of Nav1.7 against mechanical shear stress. Using molecular dynamics simulation, homology modelling and site-directed mutagenesis, we identify an intramolecular disulfide bond of ß1 (Cys21-Cys43) which is partially involved in this process: the ß1-C43A mutant prevents mechanical modulation of voltage dependence of activation, but not of fast inactivation. Our data emphasise the unique role of segment 6 of domain IV for sodium channel fast inactivation and confirm previous reports that the intracellular process of fast inactivation can be modified by interfering with the extracellular end of segment 6 of domain IV. Thus, our data suggest that physiological gating of Nav1.7 may be protected against mechanical stress in a living organism by assembly with the ß1 subunit.

Selective Ligands and Drug Discovery Targeting the Voltage-Gated Sodium Channel Nav1.7.

The voltage-gated sodium (Nav) channel Nav1.7 has been the focus of intense investigation in recent years. Human genetics studies of individuals with gain-of-function and loss-of-function mutations in the Nav1.7 channel have implicated Nav1.7 as playing a critical role in pain. Therefore, selective inhibition of Nav1.7 represents a potentially new analgesic strategy that is expected to be devoid of the significant liabilities associated with available treatment options. Although the identification and development of selective Nav channel modulators have historically been challenging, a number of recent publications has demonstrated progression of increasingly subtype-selective small molecules and peptides toward potential use in preclinical or clinical studies. In this respect, we focus on three binding sites that appear to offer the highest potential for the discovery and optimization of Nav1.7-selective inhibitors: the extracellular vestibule of the pore, the extracellular loops of voltage-sensor domain II (VSD2), and the extracellular loops of voltage-sensor domain IV (VSD4). Notably, these three receptor sites on Nav1.7 can all be defined as extracellular druggable sites, suggesting that non-small molecule formats are potential therapeutic options. In this chapter, we will review specific considerations and challenges underlying the identification and optimization of selective, potential therapeutics targeting Nav1.7 for chronic pain indications.

Selective Closed-State Nav1.7 Blocker JZTX-34 Exhibits Analgesic Effects against Pain.

Jingzhaotoxin-34 (JZTX-34) is a selective inhibitor of tetrodotoxin-sensitive (TTX-S) sodium channels. In this study, we found that JZTX-34 selectively acted on Nav1.7 with little effect on other sodium channel subtypes including Nav1.5. If the DIIS3-S4 linker of Nav1.5 is substituted by the correspond linker of Nav1.7, the sensitivity of Nav1.5 to JZTX-34 extremely increases to 1.05 µM. Meanwhile, a mutant D816R in the DIIS3-S4 linker of Nav1.7 decreases binding affinity of Nav1.7 to JZTX-34 about 32-fold. The reverse mutant R800D at the corresponding position in Nav1.5 greatly increased its binding affinity to JZTX-34. This implies that JZTX-34 binds to DIIS3-S4 linker of Nav1.7 and the critical residue of Nav1.7 is D816. Unlike ß-scorpion toxin trapping sodium channel in an open state, activity of JZTX-34 requires the sodium channel to be in a resting state. JZTX-34 exhibits an obvious analgesic effect in a rodent pain model. Especially, it shows a longer duration and is more effective than morphine in hot pain models. In a formalin-induced pain model, JZTX-34 at dose of 2 mg/kg is equipotent with morphine (5 mg/kg) in the first phase and several-fold more effective than morphine in second phase. Taken together, our data indicate that JZTX-34 releases pain by selectively binding to the domain II voltage sensor of Nav1.7 in a closed configuration.

Translational Model Systems for Complex Sodium Channel Pathophysiology in Pain.

Chronic pain patients are often left with insufficient treatment as the pathophysiology especially of neuropathic pain remains enigmatic. Recently, genetic variations in the genes of the voltage-gated sodium channels (Navs) were linked to inherited neuropathic pain syndromes, opening a research pathway to foster our understanding of the pathophysiology of neuropathic pain. More than 10 years ago, the rare, inherited pain syndrome erythromelalgia was linked to mutations in the subtype Nav1.7, and since then a plethora of mutations and genetic variations in this and other Nav genes were identified. Often the biophysical changes induced by the genetic alteration offer a straightforward explanation for the clinical symptoms, but mutations in some channels, especially Nav1.9, paint a more complex picture. Although efforts were undertaken to significantly advance our knowledge, translation from heterologous or animal model systems to humans remains a challenge. Here we present recent advances in translation using stem cell-derived human sensory neurons and their potential application for identification of better, effective, and more precise treatment for the individual pain patient.

The NAv1.7 blocker protoxin II reduces burn injury-induced spinal nociceptive processing.

Controlling pain in burn-injured patients poses a major clinical challenge. Recent findings suggest that reducing the activity of the voltage-gated sodium channel Nav1.7 in primary sensory neurons could provide improved pain control in burn-injured patients. Here, we report that partial thickness scalding-type burn injury on the rat paw upregulates Nav1.7 expression in primary sensory neurons 3 h following injury. The injury also induces upregulation in phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), a marker for nociceptive activation in primary sensory neurons. The upregulation in p-CREB occurs mainly in Nav1.7-immunopositive neurons and exhibits a peak at 5 min and, following a decline at 30 min, a gradual increase from 1 h post-injury. The Nav1.7 blocker protoxin II (ProTxII) or morphine injected intraperitoneally 15 min before or after the injury significantly reduces burn injury-induced spinal upregulation in phosphorylated serine 10 in histone H3 and phosphorylated extracellular signal-regulated kinase 1/2, which are both markers for spinal nociceptive processing. Further, ProTxII significantly reduces the frequency of spontaneous excitatory post-synaptic currents in spinal dorsal horn neurons following burn injury. Together, these findings indicate that using Nav1.7 blockers should be considered to control pain in burn injury. KEY MESSAGES: • Burn injury upregulates Nav1.7 expression in primary sensory neurons. • Burn injury results in increased activity of Nav1.7-expressing primary sensory neurons. • Inhibiting Nav1.7 by protoxin II reduces spinal nociceptive processing. • Nav1.7 represents a potential target to reduce pain in burn injury.

A Functional NaV1.7-NaVAb Chimera with a Reconstituted High-Affinity ProTx-II Binding Site.

The NaV1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel NaVAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of NaV1.7. Importantly, this chimera, DII S1-S4, forms functional sodium channels and is potently inhibited by the NaV1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [125I]ProTx-II binding and surface plasmon resonance that the purified DII S1-S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1-S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a NaV1.7-NaVAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel NaV1.7 inhibitors and for structural studies to understand the toxin-channel interaction.

Control of Neurotransmission by NaV1.7 in Human, Guinea Pig, and Mouse Airway Parasympathetic Nerves.

Little is known about the neuronal voltage-gated sodium channels (NaVs) that control neurotransmission in the parasympathetic nervous system. We evaluated the expression of the α subunits of each of the nine NaVs in human, guinea pig, and mouse airway parasympathetic ganglia. We combined this information with a pharmacological analysis of selective NaV blockers on parasympathetic contractions of isolated airway smooth muscle. As would be expected from previous studies, tetrodotoxin potently blocked the parasympathetic responses in the airways of each species. Gene expression analysis showed that that NaV 1.7 was virtually the only tetrodotoxin-sensitive NaV1 gene expressed in guinea pig and human airway parasympathetic ganglia, where mouse ganglia expressed NaV1.1, 1.3, and 1.7. Using selective pharmacological blockers supported the gene expression results, showing that blocking NaV1.7 alone can abolish the responses in guinea pig and human bronchi, but not in mouse airways. To block the responses in mouse airways requires that NaV1.7 along with NaV1.1 and/or NaV1.3 is blocked. These results may suggest novel indications for NaV1.7-blocking drugs, in which there is an overactive parasympathetic drive, such as in asthma. The data also raise the potential concern of antiparasympathetic side effects for systemic NaV1.7 blockers.

Hierarchical CRMP2 posttranslational modifications control NaV1.7 function.

Voltage-gated sodium channels are crucial determinants of neuronal excitability and signaling. Trafficking of the voltage-gated sodium channel NaV1.7 is dysregulated in neuropathic pain. We identify a trafficking program for NaV1.7 driven by hierarchical interactions with posttranslationally modified versions of the binding partner collapsin response mediator protein 2 (CRMP2). The binding described between CRMP2 and NaV1.7 was enhanced by conjugation of CRMP2 with small ubiquitin-like modifier (SUMO) and further controlled by the phosphorylation status of CRMP2. We determined that CRMP2 SUMOylation is enhanced by prior phosphorylation by cyclin-dependent kinase 5 and antagonized by Fyn phosphorylation. As a consequence of CRMP2 loss of SUMOylation and binding to NaV1.7, the channel displays decreased membrane localization and current density, and reduces neuronal excitability. Preventing CRMP2 SUMOylation with a SUMO-impaired CRMP2-K374A mutant triggered NaV1.7 internalization in a clathrin-dependent manner involving the E3 ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) and endocytosis adaptor proteins Numb and epidermal growth factor receptor pathway substrate 15. Collectively, our work shows that diverse modifications of CRMP2 cross-talk to control NaV1.7 activity and illustrate a general principle for regulation of NaV1.7.