CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Molecular and Functional Relevance of NaV1.8-Induced Atrial Arrhythmogenic Triggers in a Human SCN10A Knock-Out Stem Cell Model.

In heart failure and atrial fibrillation, a persistent Na+ current (INaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that NaV1.8 contributes to arrhythmogenesis by inducing a INaL. Genome-wide association studies indicate that mutations in the SCN10A gene (NaV1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these NaV1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the INaL and action potential duration. Ca2+ measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca2+ leak. The INaL was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of NaV1.8. No effects on atrial APD90 were detected in any groups. Both SCN10A KO and specific blockers of NaV1.8 led to decreased Ca2+ spark frequency and a significant reduction of arrhythmogenic Ca2+ waves. Our experiments demonstrate that NaV1.8 contributes to INaL formation in human atrial CMs and that NaV1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore NaV1.8 could be a new target for antiarrhythmic strategies.

Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants.

We illustrate use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia. Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M), respectively. His father showed ventricular premature beats; his mother was asymptomatic. Molecular biological characterizations demonstrated greater TNNT2 messenger RNA (mRNA) expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. Cardiac troponin Ts became progressively organized but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular Ca2+ transients, Ca2+ oscillations and increased action potential durations. SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, action potential prolongation and susceptibility to early afterdepolarizations at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Biallelic Loss of Function Mutation in Sodium Channel Gene SCN10A in an Autism Spectrum Disorder Trio from Pakistan.

The genetic dissection of autism spectrum disorders (ASD) has uncovered the contribution of de novo mutations in many single genes as well as de novo copy number variants. More recent work also suggests a strong contribution from recessively inherited variants, particularly in populations in which consanguineous marriages are common. What is also becoming more apparent is the degree of pleiotropy, whereby mutations in the same gene may have quite different phenotypic and clinical consequences. We performed whole exome sequencing in a group of 115 trios from countries with a high level of consanguineous marriages. In this paper we report genetic and clinical findings on a proband with ASD, who inherited a biallelic truncating pathogenic/likely pathogenic variant in the gene encoding voltage-gated sodium channel X alpha subunit, SCN10A (NM_006514.2:c.937G>T:(p.Gly313*)). The biallelic pathogenic/likely pathogenic variant in this study have different clinical features than heterozygous mutations in the same gene. The study of consanguineous families for autism spectrum disorder is highly valuable.

Prefrontal cortex pyramidal neurons express functional Nav1.8 tetrodotoxin-resistant sodium currents.

It has been repeatedly proved that Nav1.8 tetrodotoxin (TTX)-resistant sodium currents are expressed in peripheral sensory neurons where they play important role in nociception. There are very few publications that show the presence of TTX-resistant sodium currents in central neurons. The aim of this study was to assess if functional Nav1.8 TTX-resistant sodium currents are expressed in prefrontal cortex pyramidal neurons. All recordings were performed in the presence of TTX in the extracellular solution to block TTX-sensitive sodium currents. The TTX-resistant sodium current recorded in this study was mainly carried by the Nav1.8 sodium channel isoform because the Nav1.9 current was inhibited by the -65 mV holding potential that we used throughout the study. Moreover, the sodium current that we recorded was inhibited by treatment with the selective Nav1.8 inhibitor A-803467. Confocal microscopy experiments confirmed the presence of the Nav1.8 α subunit in prefrontal cortex pyramidal neurons. Activation and steady state inactivation properties of TTX-resistant sodium currents were also assessed in this study and they were similar to activation and inactivation properties of TTX-resistant sodium currents expressed in dorsal root ganglia (DRG) neurons. Moreover, this study showed that carbamazepine (60 µM) inhibited the maximal amplitude of the TTX-resistant sodium current. Furthermore, we found that carbamazepine shifts steady state inactivation curve of TTX-resistant sodium currents toward hyperpolarization. This study suggests that the Nav1.8 TTX-resistant sodium channel is expressed not only in DRG neurons, but also in cortical neurons and may be molecular target for antiepileptic drugs such as carbamazepine.

Protein arginine methyltransferase 7 modulates neuronal excitability by interacting with NaV1.9.

ABSTRACT: Human NaV1.9 (hNaV1.9), encoded by SCN11A, is preferentially expressed in nociceptors, and its mutations have been linked to pain disorders. NaV1.9 could be a promising drug target for pain relief. However, the modulation of NaV1.9 activity has remained elusive. Here, we identified a new candidate NaV1.9-interacting partner, protein arginine methyltransferase 7 (PRMT7). Whole-cell voltage-clamp recordings showed that coelectroporation of human SCN11A and PRMT7 in dorsal root ganglion (DRG) neurons of Scn11a-/- mice increased the hNaV1.9 current density. By contrast, a PRMT7 inhibitor (DS-437) reduced mNaV1.9 currents in Scn11a+/+ mice. Using the reporter molecule CD4, we observed an increased distribution of hLoop1 on the cell surface of PRMT7-overexpressing HKE293T cells. Furthermore, we found that PRMT7 mainly binds to residues 563 to 566 within the first intracellular loop of hNaV1.9 (hLoop1) and methylates hLoop1 at arginine residue 519. Moreover, overexpression of PRMT7 increased the number of action potential fired in DRG neurons of Scn11a+/+ mice but not Scn11a-/- mice. However, DS-437 significantly inhibited the action potential frequency of DRG neurons and relieved pain hypersensitivity in Scn11aA796G/A796G mice. In summary, our observations revealed that PRMT7 modulates neuronal excitability by regulating NaV1.9 currents, which may provide a potential method for pain treatment.

Chemogenetic modulation of sensory neurons reveals their regulating role in melanoma progression.

Sensory neurons have recently emerged as components of the tumor microenvironment. Nevertheless, whether sensory neuronal activity is important for tumor progression remains unknown. Here we used Designer Receptors Exclusively Activated by a Designer Drug (DREADD) technology to inhibit or activate sensory neurons' firing within the melanoma tumor. Melanoma growth and angiogenesis were accelerated following inhibition of sensory neurons' activity and were reduced following overstimulation of these neurons. Sensory neuron-specific overactivation also induced a boost in the immune surveillance by increasing tumor-infiltrating anti-tumor lymphocytes, while reducing immune-suppressor cells. In humans, a retrospective in silico analysis of melanoma biopsies revealed that increased expression of sensory neurons-related genes within melanoma was associated with improved survival. These findings suggest that sensory innervations regulate melanoma progression, indicating that manipulation of sensory neurons' activity may provide a valuable tool to improve melanoma patients' outcomes.

Detrimental proarrhythmogenic interaction of Ca2+/calmodulin-dependent protein kinase II and NaV1.8 in heart failure.

An interplay between Ca2+/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and late Na+ current (INaL) is known to induce arrhythmias in the failing heart. Here, we elucidate the role of the sodium channel isoform NaV1.8 for CaMKIIδc-dependent proarrhythmia. In a CRISPR-Cas9-generated human iPSC-cardiomyocyte homozygous knock-out of NaV1.8, we demonstrate that NaV1.8 contributes to INaL formation. In addition, we reveal a direct interaction between NaV1.8 and CaMKIIδc in cardiomyocytes isolated from patients with heart failure (HF). Using specific blockers of NaV1.8 and CaMKIIδc, we show that NaV1.8-driven INaL is CaMKIIδc-dependent and that NaV1.8-inhibtion reduces diastolic SR-Ca2+ leak in human failing cardiomyocytes. Moreover, increased mortality of CaMKIIδc-overexpressing HF mice is reduced when a NaV1.8 knock-out is introduced. Cellular and in vivo experiments reveal reduced ventricular arrhythmias without changes in HF progression. Our work therefore identifies a proarrhythmic CaMKIIδc downstream target which may constitute a prognostic and antiarrhythmic strategy.

Disordered breathing in a Pitt-Hopkins syndrome model involves Phox2b-expressing parafacial neurons and aberrant Nav1.8 expression.

Pitt-Hopkins syndrome (PTHS) is a rare autism spectrum-like disorder characterized by intellectual disability, developmental delays, and breathing problems involving episodes of hyperventilation followed by apnea. PTHS is caused by functional haploinsufficiency of the gene encoding transcription factor 4 (Tcf4). Despite the severity of this disease, mechanisms contributing to PTHS behavioral abnormalities are not well understood. Here, we show that a Tcf4 truncation (Tcf4tr/+) mouse model of PTHS exhibits breathing problems similar to PTHS patients. This behavioral deficit is associated with selective loss of putative expiratory parafacial neurons and compromised function of neurons in the retrotrapezoid nucleus that regulate breathing in response to tissue CO2/H+. We also show that central Nav1.8 channels can be targeted pharmacologically to improve respiratory function at the cellular and behavioral levels in Tcf4tr/+ mice, thus establishing Nav1.8 as a high priority target with therapeutic potential in PTHS.

Contribution of Multiple Inherited Variants to Autism Spectrum Disorder (ASD) in a Family with 3 Affected Siblings.

Autism Spectrum Disorder (ASD) is the most common neurodevelopmental disorder in children and shows high heritability. However, how inherited variants contribute to ASD in multiplex families remains unclear. Using whole-genome sequencing (WGS) in a family with three affected children, we identified multiple inherited DNA variants in ASD-associated genes and pathways (RELN, SHANK2, DLG1, SCN10A, KMT2C and ASH1L). All are shared among the three children, except ASH1L, which is only present in the most severely affected child. The compound heterozygous variants in RELN, and the maternally inherited variant in SHANK2, are considered to be major risk factors for ASD in this family. Both genes are involved in neuron activities, including synaptic functions and the GABAergic neurotransmission system, which are highly associated with ASD pathogenesis. DLG1 is also involved in synapse functions, and KMT2C and ASH1L are involved in chromatin organization. Our data suggest that multiple inherited rare variants, each with a subthreshold and/or variable effect, may converge to certain pathways and contribute quantitatively and additively, or alternatively act via a 2nd-hit or multiple-hits to render pathogenicity of ASD in this family. Additionally, this multiple-hits model further supports the quantitative trait hypothesis of a complex genetic, multifactorial etiology for the development of ASDs.

Common variants in SCN10A gene associated with Brugada syndrome.

Genome-wide association studies indicate that SCN10A plays an important role in cardiac electrophysiology. Common and rare SCN10A variants are suggested to contribute to Brugada Syndrome (BrS), an inherited channelopathy resulting from genetic-determined loss-of-function in cardiac sodium channel. This study sought to characterize the role of SCN10A common variants in BrS. Clinical and genetic analyses were performed in 197 patients diagnosed with BrS. Baseline ECG parameters were evaluated in patients carrying each of four common variants associated with BrS. Cellular electrophysiological study was performed in SCN5A-SCN10A co-transfected TSA201 cells to investigate the possible electrophysiological characteristics of the allele of rs6795970, which displayed the most significant association with BrS. Four SCN10A common variants (rs7630989, rs57326399, rs6795970, rs12632942) displayed significant association with BrS susceptibility. There were no evident associations between baseline ECG parameters in BrS patients and the different genotypes of the four variants. Rs6795970 (V1073) was strongly associated with a risk for BrS, which suggests the different electrophysiological characters between these two alleles. Functional study showed a positive shift in steady-state activation (V1/2: -62.2 ± 2.6 vs. -53.5 ± 1.6 for A1073 and V1073 group, respectively; P < 0.05) and slower recovery from inactivation in mutant SCN5A-SCN10A co-transfected cells with, which contribute to the slow conduction in BrS patients with rs6795970. In conclusion, SCN10A common variants are associated with increased susceptibility to BrS. An allele rs6795970 (V1073) increases the risk for BrS. The electrophysiological changes in a positive shift in steady-state activation and slower recovery from inactivation by SCN10A-V1073 contribute to this variant associated BrS.

Genetic predictors of sick sinus syndrome.

Sick sinus syndrome (SSS) encompasses a group of conduction disorders characterized by the inability of sinoatrial node to perform its pacemaker function. Our aim was to identify genetic predictors of SSS in a prospective cohort of patients admitted to the clinic for pacemaker implantation using single-locus and multilocus approaches. We performed genotyping for polymorphic markers of CLCNKA (rs10927887), SCN10A (rs6795970), FNDC3B (rs9647379), MIR146A (rs2910164), SYT10 (rs7980799), MYH6 (rs365990), and KCNE1 (rs1805127) genes in the group of 284 patients with SSS and 243 healthy individuals. Associations between the studied loci and SSS were tested using logistic regression under recessive genetic model using sex and age as covariates. Multilocus analysis was performed using Markov chain Monte Carlo method implemented in the APSampler program. Correction for multiple testing was performed using Benjamini-Hochberg procedure. We detected an individual association between KCNE1 rs1805127*A allele and SSS in the total study group (OR 0.43, PFDR = 0.028) and in the subgroup of patients with 2nd or 3rd degree sinoatrial block (OR 0.17, PFDR = 0.033), and identified seven allelic patterns associated with the disease. SCN10A rs6795970*T and MIR146A rs2910164*C alleles were present in all seven combinations associated with SSS. The highest risk of SSS was conferred by the combination SCN10A rs6795970*T+FNDC3B rs9647379*C+MIR146A rs2910164*C+SYT10 rs7980799*C+KCNE1 rs1805127*G (OR 2.98, CI 1.77-5.00, P = 1.27 × 10-5, PFDR = 0.022). Our findings suggest that KCNE1 rs1805127 polymorphism may play a role in susceptibility to sinoatrial node dysfunction, particularly presenting as 2nd or 3rd degree sinoatrial block, and the risk-modifying effect of other studied loci is better detected using multilocus approach.

Nociceptive sensory neurons promote CD8 T cell responses to HSV-1 infection.

Host protection against cutaneous herpes simplex virus 1 (HSV-1) infection relies on the induction of a robust adaptive immune response. Here, we show that Nav1.8+ sensory neurons, which are involved in pain perception, control the magnitude of CD8 T cell priming and expansion in HSV-1-infected mice. The ablation of Nav1.8-expressing sensory neurons is associated with extensive skin lesions characterized by enhanced inflammatory cytokine and chemokine production. Mechanistically, Nav1.8+ sensory neurons are required for the downregulation of neutrophil infiltration in the skin after viral clearance to limit the severity of tissue damage and restore skin homeostasis, as well as for eliciting robust CD8 T cell priming in skin-draining lymph nodes by controlling dendritic cell responses. Collectively, our data reveal an important role for the sensory nervous system in regulating both innate and adaptive immune responses to viral infection, thereby opening up possibilities for new therapeutic strategies.

Single-cell transcriptomics trajectory and molecular convergence of clinically relevant mutations in Brugada syndrome.

Brugada syndrome (BrS) is a rare, inherited arrhythmia with high risk of sudden cardiac death. To evaluate the molecular convergence of clinically relevant mutations and to identify developmental cardiac cell types that are associated with BrS etiology, we collected 733 mutations represented by 16 sodium, calcium, potassium channels, and regulatory and structural genes related to BrS. Among the clinically relevant mutations, 266 are unique singletons and 88 mutations are recurrent. We observed an over-representation of clinically relevant mutations (∼80%) in SCN5A gene and also identified several candidate genes, including GPD1L, TRPM4, and SCN10A. Furthermore, protein domain enrichment analysis revealed that a large proportion of the mutations impacted ion transport domains in multiple genes, including SCN5A, TRPM4, and SCN10A. A comparative protein domain analysis of SCN5A further established a significant (P = 0.04) enrichment of clinically relevant mutations within ion transport domain, including a significant (P = 0.02) mutation hotspot within 1321-1380 residue. The enrichment of clinically relevant mutations within SCN5A ion transport domain is stronger (P = 0.00003) among early onset of BrS. Our spatiotemporal cellular heart developmental (prenatal to adult) trajectory analysis applying single-cell transcriptome identified the most frequently BrS-mutated genes (SCN5A and GPD1L) are significantly upregulated in the prenatal cardiomyocytes. A more restrictive cellular expression trajectory is prominent in the adult heart ventricular cardiomyocytes compared to prenatal. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS-associated cell type that provides insight into the complex genetic etiology of BrS.NEW & NOTEWORTHY Brugada syndrome is a rare inherited arrhythmia with high risk of sudden cardiac death. We present the findings for a molecular convergence of clinically relevant mutations and identification of a single-cell transcriptome-derived cardiac cell types that are associated with the etiology of BrS. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS-associated cell type that provides insight into the complex genetic etiology of BrS.

Variant Intronic Enhancer Controls SCN10A-short Expression and Heart Conduction.

BACKGROUND: Genetic variants in SCN10A, encoding the neuronal voltage-gated sodium channel NaV1.8, are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities, and heart rate. The cardiac function of SCN10A has not been resolved, however, and diverging mechanisms have been proposed. Here, we investigated the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A, encoding the major essential cardiac sodium channel NaV1.5. METHODS: The expression of SCN10A was investigated in mouse and human hearts. With the use of CRISPR/Cas9 genome editing, the mouse intronic enhancer was disrupted, and mutant mice were characterized by transcriptomic and electrophysiological analyses. The association of genetic variants at SCN5A-SCN10A enhancer regions and gene expression were evaluated by genome-wide association studies single-nucleotide polymorphism mapping and expression quantitative trait loci analysis. RESULTS: We found that cardiomyocytes of the atria, sinoatrial node, and ventricular conduction system express a short transcript comprising the last 7 exons of the gene (Scn10a-short). Transcription occurs from an intronic enhancer-promoter complex, whereas full-length Scn10a transcript was undetectable in the human and mouse heart. Expression quantitative trait loci analysis revealed that the genetic variants in linkage disequilibrium with genetic variant rs6801957 in the intronic enhancer associate with SCN10A transcript levels in the heart. Genetic modification of the enhancer in the mouse genome led to reduced cardiac Scn10a-short expression in atria and ventricles, reduced cardiac sodium current in atrial cardiomyocytes, atrial conduction slowing and arrhythmia, whereas the expression of Scn5a, the presumed enhancer target gene, remained unaffected. In patch-clamp transfection experiments, expression of Scn10a-short-encoded NaV1.8-short increased NaV1.5-mediated sodium current. We propose that noncoding genetic variation modulates transcriptional regulation of Scn10a-short in cardiomyocytes that impacts NaV1.5-mediated sodium current and heart rhythm. CONCLUSIONS: Genetic variants in and around SCN10A modulate enhancer function and expression of a cardiac-specific SCN10A-short transcript. We propose that noncoding genetic variation modulates transcriptional regulation of a functional C-terminal portion of NaV1.8 in cardiomyocytes that impacts on NaV1.5 function, cardiac conduction velocities, and arrhythmia susceptibility.

Pain behavior in SCN9A (Nav1.7) and SCN10A (Nav1.8) mutant rodent models.

The two voltage gated sodium channels Nav1.7 and Nav1.8 are expressed in the peripheral nervous system and involved in various pain conditions including inflammatory and neuropathic pain. Rodent models bearing deletions or mutations of the corresponding genes, Scn9a and Scn10a, were created in order to understand the role of these channels in the pathophysiological mechanism underlying pain symptoms. This review summarizes the pain behavior profiles reported in Scn9a and Scn10a rodent models. The complete loss-of-function or knockout (KO) of Scn9a or Scn10a and the conditional KO (cKO) of Scn9a in specific cell populations were shown to decrease sensitivity to various pain stimuli. The Possum mutant mice bearing a dominant hypermorphic mutation in Scn10a revealed higher sensitivity to noxious stimuli. Several gain-of-function mutations were identified in patients with painful small fiber neuropathy. Future knowledge obtained from preclinical models bearing these mutations will allow understanding how these mutations affect pain. In addition, the review gives perspectives for creating models that better mimic patients' pain symptoms in view to developing novel analgesic strategies.

MicroRNA-96 is required to prevent allodynia by repressing voltage-gated sodium channels in spinal cord.

Voltage-gated sodium channels (Navs) 1.7, 1.8, and 1.9 are predominately expressed in peripheral sensory neurons and are critical for action potential propagation in nociceptors. Unexpectedly, we found that expression of SCN9A, SCN10A, SCN11A, and SCN2A, the alpha subunit of Nav1.7, Nav1.8, Nav1.9 and Nav1.2, respectively, are up-regulated in spinal dorsal horn (SDH) neurons of miR-96 knockout mice. These mice also have de-repression of CACNA2D1/2 in DRG and display thermal and mechanical allodynia that could be attenuated by intrathecal or intraperitoneal injection of Nav1.7 or Nav1.8 blockers or Gabapentin. Moreover, Gad2::CreERT2 conditional miR-96 knockout mice phenocopied global knockout mice, implicating inhibitory neurons; nerve injury induced significant loss of miR-96 in SDH GABAergic and Glutamatergic neurons in mice which negatively correlated to up-regulation of Nav1.7, Nav1.8, Nav1.9 and Scn2a, this dis-regulation of miR-96 and Navs in SDH neurons contributed to neuropathic pain which can be alleviated by intrathecal injection of Nav1.7 or Nav1.8 blockers. In conclusion, miR-96 is required to avoid allodynia through limiting the expression of VGCCs and Navs in DRG and Navs in SDH in naïve and nerve injury-induced neuropathic pain mice. Our findings suggest that central nervous system penetrating Nav1.7 and Nav1.8 blockers may be efficacious for pain relief.

Antinociceptive properties of an isoform-selective inhibitor of Nav1.7 derived from saxitoxin in mouse models of pain.

ABSTRACT: The voltage-gated sodium channel Nav1.7 is highly expressed in nociceptive afferents and is critically involved in pain signal transmission. Nav1.7 is a genetically validated pain target in humans because loss-of-function mutations cause congenital insensitivity to pain and gain-of-function mutations cause severe pain syndromes. Consequently, pharmacological inhibition has been investigated as an analgesic therapeutic strategy. We describe a small molecule Nav1.7 inhibitor, ST-2530, that is an analog of the naturally occurring sodium channel blocker saxitoxin. When evaluated against human Nav1.7 by patch-clamp electrophysiology using a protocol that favors the resting state, the Kd of ST-2530 was 25 ± 7 nM. ST-2530 exhibited greater than 500-fold selectivity over human voltage-gated sodium channel isoforms Nav1.1-Nav1.6 and Nav1.8. Although ST-2530 had lower affinity against mouse Nav1.7 (Kd = 250 ± 40 nM), potency was sufficient to assess analgesic efficacy in mouse pain models. A 3-mg/kg dose administered subcutaneously was broadly analgesic in acute pain models using noxious thermal, mechanical, and chemical stimuli. ST-2530 also reversed thermal hypersensitivity after a surgical incision on the plantar surface of the hind paw. In the spared nerve injury model of neuropathic pain, ST-2530 transiently reversed mechanical allodynia. These analgesic effects were demonstrated at doses that did not affect locomotion, motor coordination, or olfaction. Collectively, results from this study indicate that pharmacological inhibition of Nav1.7 by a small molecule agent with affinity for the resting state of the channel is sufficient to produce analgesia in a range of preclinical pain models.