A central mechanism of analgesia in mice and humans lacking the sodium channel NaV1.7.
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ABSTRACT:
SUBMITTER: MacDonald DI
PROVIDER: S-EPMC8110947 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background and purposeThe development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Nav 1.1-Nav 1.7.Experimental approachEach VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings.Key resultsThe inhibitory effects of TTX on Nav 1.1-Nav 1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Nav 1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Nav 1.7, Thr1425 and Ile1426 were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Nav 1.7 T1425M and Nav 1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively.Conclusions and implicationsThe reduced binding of chiriquitoxin to Nav 1.7 was attributed to its C11-OH and/or C12-NH2 , based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.
| S-EPMC5647187 | biostudies-literature
Project description:The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.
| S-EPMC9325523 | biostudies-literature
Project description:Gating modifier toxins (GMTs) isolated from venomous organisms such as Protoxin-II (ProTx-II) and Huwentoxin-IV (HwTx-IV) that inhibit the voltage-gated sodium channel NaV1.7 by binding to its voltage-sensing domain II (VSDII) have been extensively investigated as non-opioid analgesics. However, reliably predicting how a mutation to a GMT will affect its potency for NaV1.7 has been challenging. Here, we hypothesize that structure-based computational methods can be used to predict such changes. We employ free-energy perturbation (FEP), a physics-based simulation method for predicting the relative binding free energy (RBFE) between molecules, and the cryo electron microscopy (cryo-EM) structures of ProTx-II and HwTx-IV bound to VSDII of NaV1.7 to re-predict the relative potencies of forty-seven point mutants of these GMTs for NaV1.7. First, FEP predicted these relative potencies with an overall root mean square error (RMSE) of 1.0 ± 0.1 kcal/mol and an R2 value of 0.66, equivalent to experimental uncertainty and an improvement over the widely used molecular-mechanics/generalized born-surface area (MM-GB/SA) RBFE method that had an RMSE of 3.9 ± 0.8 kcal/mol. Second, inclusion of an explicit membrane model was needed for the GMTs to maintain stable binding poses during the FEP simulations. Third, MM-GB/SA and FEP were used to identify fifteen non-standard tryptophan mutants at ProTx-II[W24] predicted in silico to have a at least a 1 kcal/mol gain in potency. These predicted potency gains are likely due to the displacement of high-energy waters as identified by the WaterMap algorithm for calculating the positions and thermodynamic properties of water molecules in protein binding sites. Our results expand the domain of applicability of FEP and set the stage for its prospective use in biologics drug discovery programs involving GMTs and NaV1.7.
| S-EPMC8002187 | biostudies-literature
Project description:Several distinct components of voltage-gated sodium current have been recorded from native dorsal root ganglion (DRG) neurons that display differences in gating and pharmacology. This study compares the electrophysiological properties of two peripheral nerve sodium channels that are expressed selectively in DRG neurons (Na(v)1.7 and Na(v)1.8). Recombinant Na(v)1.7 and Na(v)1.8 sodium channels were coexpressed with the auxiliary beta(1) subunit in Xenopus oocytes. In this system coexpression of the beta(1) subunit with Na(v)1.7 and Na(v)1.8 channels results in more rapid inactivation, a shift in midpoints of steady-state activation and inactivation to more hyperpolarizing potentials, and an acceleration of recovery from inactivation. The coinjection of beta(1) subunit also significantly increases the expression of Na(v)1.8 by sixfold but has no effect on the expression of Na(v)1.7. In addition, a great percentage of Na(v)1.8+beta(1) channels is observed to enter rapidly into the slow inactivated states, in contrast to Nav1.7+beta(1) channels. Consequently, the rapid entry into slow inactivation is believed to cause a frequency-dependent reduction of Na(v)1.8+beta(1) channel amplitudes, seen during repetitive pulsing between 1 and 2 Hz. However, at higher frequencies (>20 Hz) Na(v)1.8+beta(1) channels reach a steady state to approximately 42% of total current. The presence of this steady-state sodium channel activity, coupled with the high activation threshold (V(0.5) = -3.3 mV) of Na(v)1.8+beta(1), could enable the nociceptive fibers to fire spontaneously after nerve injury.
| S-EPMC6763856 | biostudies-literature
Project description: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, β-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.
| S-EPMC7012836 | biostudies-literature
Project description:Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder often accompanied by intellectual disability, language impairment and medical co-morbidities. The heritability of autism is high and multiple genes have been implicated as causal. However, most of these genes have been identified in de novo cases. To further the understanding of familial autism, we performed whole-exome sequencing on five families in which second- and third-degree relatives were affected. By focusing on novel and protein-altering variants, we identified a small set of candidate genes. Among these, a novel private missense C1143F variant in the second intracellular loop of the voltage-gated sodium channel NaV1.7, encoded by the SCN9A gene, was identified in one family. Through electrophysiological analysis, we show that NaV1.7C1143F exhibits partial loss-of-function effects, resulting in slower recovery from inactivation and decreased excitability in cultured cortical neurons. Furthermore, for the same intracellular loop of NaV1.7, we found an excess of rare variants in a case-control variant-burden study. Functional analysis of one of these variants, M932L/V991L, also demonstrated reduced firing in cortical neurons. However, although this variant is rare in Caucasians, it is frequent in Latino population, suggesting that genetic background can alter its effects on phenotype. Although the involvement of the SCN1A and SCN2A genes encoding NaV1.1 and NaV1.2 channels in de novo ASD has previously been demonstrated, our study indicates the involvement of inherited SCN9A variants and partial loss-of-function of NaV1.7 channels in the etiology of rare familial ASD.
| S-EPMC5468514 | biostudies-literature
Project description: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.
| S-EPMC8043720 | biostudies-literature
Project description:Loss-of-function variants in the gene SCN2A, which encodes the sodium channel NaV1.2, are strongly associated with autism spectrum disorder and intellectual disability. An estimated 20%-30% of children with these variants also suffer from epilepsy, with altered neuronal activity originating in neocortex, a region where NaV1.2 channels are expressed predominantly in excitatory pyramidal cells. This is paradoxical, as sodium channel loss in excitatory cells would be expected to dampen neocortical activity rather than promote seizure. Here, we examined pyramidal neurons lacking NaV1.2 channels and found that they were intrinsically hyperexcitable, firing high-frequency bursts of action potentials (APs) despite decrements in AP size and speed. Compartmental modeling and dynamic-clamp recordings revealed that NaV1.2 loss prevented potassium channels from properly repolarizing neurons between APs, increasing overall excitability by allowing neurons to reach threshold for subsequent APs more rapidly. This cell-intrinsic mechanism may, therefore, account for why SCN2A loss-of-function can paradoxically promote seizure.
| S-EPMC8719649 | biostudies-literature
Project description:Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids.
| S-EPMC4686868 | biostudies-literature
Project description:Loss-of-function mutations in NaV1.7 cause congenital insensitivity to pain (CIP); this voltage-gated sodium channel is therefore a key target for analgesic drug development. Utilizing a multi-modal approach, we investigated how NaV1.7 mutations lead to human pain insensitivity. Skin biopsy and microneurography revealed an absence of C-fiber nociceptors in CIP patients, reflected in a reduced cortical response to capsaicin on fMRI. Epitope tagging of endogenous NaV1.7 revealed the channel to be localized at the soma membrane, axon, axon terminals, and the nodes of Ranvier of induced pluripotent stem cell (iPSC) nociceptors. CIP patient-derived iPSC nociceptors exhibited an inability to properly respond to depolarizing stimuli, demonstrating that NaV1.7 is a key regulator of excitability. Using this iPSC nociceptor platform, we found that some NaV1.7 blockers undergoing clinical trials lack specificity. CIP, therefore, arises due to a profound loss of functional nociceptors, which is more pronounced than that reported in rodent models, or likely achievable following acute pharmacological blockade. VIDEO ABSTRACT.
| S-EPMC6424805 | biostudies-literature