Schwann cell-secreted PGE2 promotes sensory neuron excitability during development.

Electrical excitability-the ability to fire and propagate action potentials-is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage-gated sodium channels, and to fire action potential trains. Inactivating this signaling pathway in Schwann cells impairs somatosensory neuron maturation, causing multimodal sensory defects that persist into adulthood. Collectively, our studies uncover a neurodevelopmental role for prostaglandin E2 distinct from its established role in inflammation, revealing a cell non-autonomous mechanism by which glia regulate neuronal excitability to enable the development of normal sensory functions.

Structure of the Nav1.4-ß1 Complex from Electric Eel.

Voltage-gated sodium (Nav) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNav1.4, the Nav channel from electric eel, in complex with the ß1 subunit at 4.0 Å resolution. The immunoglobulin domain of ß1 docks onto the extracellular L5I and L6IV loops of EeNav1.4 via extensive polar interactions, and the single transmembrane helix interacts with the third voltage-sensing domain (VSDIII). The VSDs exhibit "up" conformations, while the intracellular gate of the pore domain is kept open by a digitonin-like molecule. Structural comparison with closed NavPaS shows that the outward transfer of gating charges is coupled to the iris-like pore domain dilation through intricate force transmissions involving multiple channel segments. The IFM fast inactivation motif on the III-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in repeats III and IV, suggesting a potential allosteric blocking mechanism for fast inactivation.

A tamoxifen receptor within a voltage-gated sodium channel.

Voltage-gated sodium channels are targets for many analgesic and antiepileptic drugs whose therapeutic mechanisms and binding sites have been well characterized. We describe the identification of a previously unidentified receptor site within the NavMs voltage-gated sodium channel. Tamoxifen, an estrogen receptor modulator, and its primary and secondary metabolic products bind at the intracellular exit of the channel, which is a site that is distinct from other previously characterized sodium channel drug sites. These compounds inhibit NavMs and human sodium channels with similar potencies and prevent sodium conductance by delaying channel recovery from the inactivated state. This study therefore not only describes the structure and pharmacology of a site that could be leveraged for the development of new drugs for the treatment of sodium channelopathies but may also have important implications for off-target health effects of this widely used therapeutic drug.

Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore region.

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.

Development and characterization of the mode-of-action of inhibitory and agonist peptides targeting the voltage-gated sodium channel SCN1B beta-subunit.

Cardiac arrhythmia treatment is a clinical challenge necessitating safer and more effective therapies. Recent studies have highlighted the role of the perinexus, an intercalated disc nanodomain enriched in voltage-gated sodium channels including both Nav1.5 and ß1 subunits, adjacent to gap junctions. These findings offer insights into action potential conduction in the heart. A 19-amino acid SCN1B (ß1/ß1B) mimetic peptide, ßadp1, disrupts VGSC beta subunit-mediated adhesion in cardiac perinexii, inducing arrhythmogenic changes. We aimed to explore ßadp1's mechanism and develop novel SCN1B mimetic peptides affecting ß1-mediated adhesion. Using patch clamp assays in neonatal rat cardiomyocytes and electric cell substrate impedance sensing (ECIS) in ß1-expressing cells, we observed ßadp1 maintained inhibitory effects for up to 5 h. A shorter peptide (LQLEED) based on the carboxyl-terminus of ßadp1 mimicked this inhibitory effect, while dimeric peptides containing repeated LQLEED sequences paradoxically promoted intercellular adhesion over longer time courses. Moreover, we found a link between these peptides and ß1-regulated intramembrane proteolysis (RIP) - a signaling pathway effecting gene transcription including that of VGSC subunits. ßadp1 increased RIP continuously over 48 h, while dimeric agonists acutely boosted RIP for up to 6 h. In the presence of DAPT, an RIP inhibitor, ßadp1's effects on ECIS-measured intercellular adhesion was reduced, suggesting a relationship between RIP and the peptide's inhibitory action. In conclusion, novel SCN1B (ß1/ß1B) mimetic peptides are reported with the potential to modulate intercellular VGSC ß1-mediated adhesion, potentially through ß1 RIP. These findings suggest a path towards the development of anti-arrhythmic drugs targeting the perinexus.

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.

Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal subunit regulation of the cardiac voltage-gated sodium channel.

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory ß-subunits. The ß-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous ß-subunits. To better reveal ß-subunit functions, we engineered a human cell line devoid of endogenous NaV ß-subunits and their immediate phylogenetic relatives. This new cell line, ß-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the ß-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to ß-subunit modulation and revealed that each ß-subunit and even MPZ imparted unique gating properties. Furthermore, combining ß1 and ß2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of ß-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/ß NaV complexes.

Variant-specific in vitro neuronal network phenotypes and drug sensitivity in SCN2A developmental and epileptic encephalopathy.

De novo variants in the NaV1.2 voltage-gated sodium channel gene SCN2A are among the major causes of developmental and epileptic encephalopathies (DEE). Based on their biophysical impact on channel conductance and gating, SCN2A DEE variants can be classified into gain-of-function (GoF) or loss-of-function (LoF). Clinical and functional data have linked early seizure onset DEE to the GoF SCN2A variants, whereas late seizure onset DEE is associated with the loss of SCN2A function. This study aims to assess the impact of GoF and LoF SCN2A variants on cultured neuronal network activity and explore their modulation by selected antiseizure medications (ASM). To this end, primary cortical cultures were generated from two knock-in mouse lines carrying variants corresponding to human GoF SCN2A p.R1882Q and LoF p.R853Q DEE variant. In vitro neuronal network activity and responses to ASM were analyzed using multielectrode array (MEA) between 2 and 4 weeks in culture. The SCN2A p.R1882Q neuronal cultures showed significantly greater mean firing and burst firing. Their network synchronicity was also higher. In contrast, the SCN2A p.R853Q cultures showed lower mean firing rate, and burst firing events were less frequent. The network synchronicity was also lower. Phenytoin and levetiracetam reduced the excitability of GoF cultures, while retigabine showed differential and potentially beneficial effects on cultures with both GoF and LoF variants. We conclude that in vitro neuronal networks harboring SCN2A GoF or LoF DEE variants present with distinctive phenotypes and responses to ASM.

Inhibition of TTX-S Na+ currents by a novel blocker QLS-278 for antinociception.

Genetic loss-of-function mutations of Nav1.7 channel, abundantly expressed in peripheral nociceptive neurons, cause congenital insensitivity to pain (CIP) in humans, indicating that selective inhibition of the channel may lead to potential therapy of pain disorders. In this study, we investigated a novel compound, 5-chloro-N-(cyclopropylsulfonyl)-2-fluoro-4-(2-(8-(furan-2-ylmethyl)-8-azaspiro [4.5] decan-2-yl) ethoxy) benzamide (QLS-278) that inhibits Nav1.7 channel and exhibits anti-nociceptive activity. Compound QLS-278 exhibits inactivation- and concentration-dependent inhibition of macroscopic currents of Nav1.7 channels stably expressed in HEK293 cells with an IC50 of 1.2 {plus minus} 0.2 µM. QLS-278 causes a hyperpolarization shift of the channel inactivation and delays recovery from inactivation, without an obvious effect on voltage-dependent activation. In mouse DRG neurons, QLS-278 suppresses native TTX-sensitive Nav currents and also reduces neuronal firing. Moreover, QLS-278 dose-dependently relieves neuropathic pain induced by spared nerve injury and inflammatory pain induced by formalin without significant alteration of spontaneous locomotor activity in mice. Altogether, our identification of the novel compound QLS-278 may hold developmental potential for the treatment of chronic pain. Significance Statement QLS-278, a novel voltage-gated sodium Nav1.7 channel blocker, inhibits native TTX-S Na+ current and reduces action potential firings in DRG sensory neurons. QLS-278 also exhibits antinociceptive activity in mouse models of pain, thus demonstrating potential for the development of a treatment for chronic pain.

Toxic to the touch: The makings of lethal mantles in pitohui birds and poison dart frogs.

How do chemically defended animals resist their own toxins? This intriguing question on the concept of autotoxicity is at the heart of how species interactions evolve. In this issue of Molecular Ecology (Molecular Ecology, 2024, 33), Bodawatta and colleagues report on how Papua New Guinean birds coopted deadly neurotoxins to create lethal mantles that protect against predators and parasites. Combining chemical screening of the plumage of a diverse collection of passerine birds with genome sequencing, the researchers unlocked a deeper understanding of how some birds sequester deadly batrachotoxin (BTX) from their food without poisoning themselves. They identified that birds impervious to BTX bear amino acid substitutions in the toxin-binding site of the voltage-gated sodium channel Nav1.4, whose function is essential for proper contraction and relaxation of vertebrate muscles. Comparative genetic and molecular docking analyses show that several of the substitutions associated with insensitivity to BTX may have become prevalent among toxic birds through positive selection. Intriguingly, poison dart frogs that also co-opted BTX in their lethal mantles were found to harbour similar toxin insensitivity substitutions in their Nav1.4 channels. Taken together, this sets up a powerful model system for studying the mechanisms behind convergent molecular evolution and how it may drive biological diversity.

Voluntary running wheel exercise induces cognitive improvement post traumatic brain injury in mouse model through redressing aberrant excitation regulated by voltage-gated sodium channels 1.1, 1.3, and 1.6.

Traumatic brain injury (TBI) leads to disturbed brain discharge rhythm, elevated excitability, anxiety-like behaviors, and decreased learning and memory capabilities. Cognitive dysfunctions severely affect the quality of life and prognosis of TBI patients, requiring effective rehabilitation treatment. Evidence indicates that moderate exercise after brain injury decreases TBI-induced cognitive decline. However, the underlying mechanism remains unelucidated. Our results demonstrate that TBI causes cognitive impairment behavior abnormalities and overexpression of Nav1.1, Nav1.3 and Nav1.6 proteins inside the hippocampus of mice models. Three weeks of voluntary running wheel (RW) exercise treatments before or/and post-injury effectively redressed the aberrant changes caused by TBI. Additionally, a 10% exercise-conditioned medium helped recover cell viability, neuronal sodium current and expressions of Nav1.1, Nav1.3 and Nav1.6 proteins across cultured neurons after injury. Therefore, the results validate the neuroprotection induced by voluntary RW exercise treatment before or/and post-TBI. The RW exercise-induced improvement in cognitive behaviors and neuronal excitability could be associated with correcting the Nav1.1, Nav1.3, and Nav1.6 expression levels. The current study proves that voluntary exercise is an effective treatment strategy against TBI. The study also highlights novel potential targets for rehabilitating TBI, including the Navs proteins.

Crosstalk among WEE1 Kinase, AKT, and GSK3 in Nav1.2 Channelosome Regulation.

The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.

Functional Characteristics of the Nav1.1 p.Arg1596Cys Mutation Associated with Varying Severity of Epilepsy Phenotypes.

Mutations of the SCN1A gene, which encodes the voltage-dependent Na+ channel's α subunit, are associated with diverse epileptic syndromes ranging in severity, even intra-family, from febrile seizures to epileptic encephalopathy. The underlying cause of this variability is unknown, suggesting the involvement of additional factors. The aim of our study was to describe the properties of mutated channels and investigate genetic causes for clinical syndromes' variability in the family of five SCN1A gene p.Arg1596Cys mutation carriers. The analysis of additional genetic factors influencing SCN1A-associated phenotypes was conducted through exome sequencing (WES). To assess the impact of mutations, we used patch clamp analysis of mutated channels expressed in HEK cells and in vivo neural excitability studies (NESs). In cells expressing the mutant channel, sodium currents were reduced. NESs indicated increased excitability of peripheral motor neurons in mutation carriers. WES showed the absence of non-SCA1 pathogenic variants that could be causative of disease in the family. Variants of uncertain significance in three genes, as potential modifiers of the most severe phenotype, were identified. The p.Arg1596Cys substitution inhibits channel function, affecting steady-state inactivation kinetics. Its clinical manifestations involve not only epileptic symptoms but also increased excitability of peripheral motor fibers. The role of Nav1.1 in excitatory neurons cannot be ruled out as a significant factor of the clinical phenotype.

Effects of Ranolazine and Ivabradine on Pulmonary Microhemodynamics in Experimental Model of Pulmonary Thromboembolism.

We studied changes of pulmonary microhemodynamics when modeling pulmonary artery thromboembolism on perfused isolated rabbit lungs after pretreatment with ranolazine and ivabradine. The increase in pulmonary artery pressure, pulmonary vascular resistance, and pre- and postcapillary resistance was less pronounced than in control animals, but was close to that in case of pulmonary thromboembolism after pretreatment with voltage-gated Na+ channel blockers lidocaine and ropivacaine. The increase of capillary filtration coefficient inversely correlated with values of capillary hydrostatic pressure. Thus, ranolazine and ivabradine exhibit the properties of voltage-gated Na+ channel blockers mainly in smooth muscles of pulmonary arterial vessels and promote the decrease in endothelial permeability.

Structural and functional insights into the inhibition of human voltage-gated sodium channels by µ-conotoxin KIIIA disulfide isomers.

µ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (NaV) subtypes, including NaV1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a 'native' CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, µ-conotoxin KIIIA, the smallest and most studied µ-conotoxin with inhibitory activity at NaV1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native µ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native µ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three µ-conotoxin KIIIA isomers displaying distinct bioactivity and NaV subtype selectivity across human NaV channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits NaV subtypes with a rank order of potency of NaV1.4 > 1.2 > 1.7 and Isomer 2 in the order of NaV1.4≈1.2 > 1.7, while the native isomer inhibited NaV1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNaV1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of µ-conotoxins targeting therapeutically relevant NaV subtypes.

Afferent neurons of the kidney with impaired firing pattern in inflammation - role of sodium currents?

Peripheral neurons with renal afferents exhibit a predominantly tonic firing pattern of higher frequency that is reduced to low frequencies (phasic firing pattern) in renal inflammation. We wanted to test the hypothesis that the reduction in firing activity during inflammation is due to high-activity tonic neurons switching from higher to low frequencies depending on altered sodium currents. We identified and cultivated afferent sensory neurons with renal projections from the dorsal root ganglia (Th11-L2). Cultivated neurons were incubated with the chemokine CXCL1 (1,5 nmol/ml) for 12 h. We characterized neurons as "tonic," i.e., sustained action potential (AP) firing, or "phasic," i.e., < 5 APs upon stimulation in the current clamp. Their membrane currents were investigated in a voltage clamp. Data analyzed: renal vs. non-renal and tonic vs. phasic neurons. Renal afferent neurons exposed to CXCL1 showed a decrease in tonic firing pattern (CXCL1: 35,6% vs. control: 57%, P < 0.05). Na+ and K+ currents were not different between control renal and non-renal DRG neurons. Phasic neurons exhibited higher Na+ and K+ currents than tonic resulting in shorter APs (3.7 ± 0.3 vs. 6.1 ± 0.6 ms, P < 0.01). In neurons incubated with CXCL1, Na+ and K+ peak current density increased in phasic (Na+: - 969 ± 47 vs. - 758 ± 47 nA/pF, P < 0.01; K+: 707 ± 22 vs. 558 ± 31 nA/pF, P < 0.01), but were unchanged in tonic neurons. Phasic neurons exposed to CXCL1 showed a broader range of Na+ currents ([- 365- - 1429 nA] vs. [- 412- - 4273 nA]; P < 0.05) similar to tonic neurons. After CXCL1 exposure, significant changes in phasic neurons were observed in sodium activation/inactivation as well as a wider distribution of Na+ currents characteristic of tonic neurons. These findings indicate a subgroup of tonic neurons besides mere tonic or phasic neurons exists able to exhibit a phasic activity pattern under pathological conditions.

Action potential conduction in the mouse and rat vagus nerve is dependent on multiple voltage-gated sodium channels (NaV1s).

Action potential (AP) conduction depends on voltage-gated sodium channels, of which there are nine subtypes. The vagus nerve, comprising sensory afferent fibers and efferent parasympathetic fibers, provides autonomic regulation of visceral organs, but the voltage-gated sodium channels (NaV1) subtypes involved in its AP conduction are poorly defined. We studied the A- and C-waves of electrically stimulated compound action potentials (CAPs) of the mouse and rat vagus nerves with and without NaV1 inhibitor administration: tetrodotoxin (TTX), PF-05089771 (mouse NaV1.7), ProTX-II (NaV1.7), ICA-121341 (NaV1.1, NaV1.3, and NaV1.6), LSN-3049227 (NaV1.2, NaV1.6, and NaV1.7), and A-803467 (NaV1.8). We show that TTX-sensitive NaV1 channels are essential for all vagal AP conduction. PF-05089771 but not ICA-121341 inhibited the mouse A-wave, which was abolished by LSN-3049227, suggesting roles for NaV1.7 and NaV1.2. The mouse C-wave was abolished by LSN-3049227 and a combination of PF-05089771 and ICA-121341, suggesting roles for NaV1.7 and NaV1.6. The rat A-wave was inhibited by ProTX-II, ICA-121341, and a combination of these inhibitors but only abolished by LSN-3049227, suggesting roles for NaV1.7, NaV1.6, and NaV1.2. The rat C-wave was abolished by LSN-3049227 and a combination of ProTX-II and ICA-121341, suggesting roles for NaV1.7 and NaV1.6. A-803467 also inhibited the mouse and rat CAP suggesting a cooperative role for the TTX-resistant NaV1.8. Overall, our data demonstrate that multiple NaV1 subtypes contribute to vagal CAPs, with NaV1.7 and NaV1.8 playing predominant roles and NaV1.6 and NaV1.2 contributing to a different extent based on nerve fiber type and species. Inhibition of these NaV1 may impact autonomic regulation of visceral organs.NEW & NOTEWORTHY Distinct NaV1 channels are involved in action potential (AP) initiation and conduction from afferent terminals within specific organs. Here, we have identified the NaV1 necessary for AP conduction in the entire murine and rat vagus nerve. We show TTX-sensitive channels are essential for all AP conduction, predominantly NaV1.7 with NaV1.2 and NaV1.6 playing lesser roles depending on the species and fiber type. In addition, we show that NaV1.8 is also essential for most axonal AP conduction.

The voltage-gated sodium channel NaV 1.7 underlies endometriosis-associated chronic pelvic pain.

Chronic pelvic pain (CPP) is the primary symptom of endometriosis patients, but adequate treatments are lacking. Modulation of ion channels expressed by sensory nerves innervating the viscera has shown promise for the treatment of irritable bowel syndrome and overactive bladder. However, similar approaches for endometriosis-associated CPP remain underdeveloped. Here, we examined the role of the voltage-gated sodium (NaV ) channel NaV 1.7 in (i) the sensitivity of vagina-innervating sensory afferents and investigated whether (ii) NaV 1.7 inhibition reduces nociceptive signals from the vagina and (iii) ameliorates endometriosis-associated CPP. The mechanical responsiveness of vagina-innervating sensory afferents was assessed with ex vivo single-unit recording preparations. Pain evoked by vaginal distension (VD) was quantified by the visceromotor response (VMR) in vivo. In control mice, pharmacological activation of NaV 1.7 with OD1 sensitised vagina-innervating pelvic afferents to mechanical stimuli. Using a syngeneic mouse model of endometriosis, we established that endometriosis sensitised vagina-innervating pelvic afferents to mechanical stimuli. The highly selective NaV 1.7 inhibitor Tsp1a revealed that this afferent hypersensitivity occurred in a NaV 1.7-dependent manner. Moreover, in vivo intra-vaginal treatment with Tsp1a reduced the exaggerated VMRs to VD which is characteristic of mice with endometriosis. Conversely, Tsp1a did not alter ex vivo afferent mechanosensitivity nor in vivo VMRs to VD in Sham control mice. Collectively, these findings suggest that NaV 1.7 plays a crucial role in endometriosis-induced vaginal hyperalgesia. Importantly, NaV 1.7 inhibition selectively alleviated endometriosis-associated CPP without the loss of normal sensation, suggesting that selective targeting of NaV 1.7 could improve the quality of life of women with endometriosis.

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing.

PURPOSE: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains below 50%, suggesting that clinically relevant variants may be missed by standard analyses. Here, we analyze "poison exons" (PEs), which are evolutionarily conserved alternative exons often absent from standard gene annotations. Variants that alter PE inclusion can lead to loss of function and may be highly penetrant contributors to disease. METHODS: We curated published RNA sequencing data from developing mouse cortex to define 1937 conserved PE regions potentially relevant to NDDs, and we analyzed variants found by genome sequencing in multiple NDD cohorts. RESULTS: Across 2999 probands, we found 6 novel clinically relevant variants in PE regions. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family (SCN1A, SCN2A, and SCN8A), which is associated with epilepsies. One variant is in SNRPB, associated with cerebrocostomandibular syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and in genes with gene-phenotype associations consistent with each probands reported features. CONCLUSION: With a very minimal increase in variant analysis burden (average of 0.77 variants per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

Multiple mutations in the Nav1.4 sodium channel of New Guinean toxic birds provide autoresistance to deadly batrachotoxin.

Toxicity has evolved multiple times across the tree of life and serves important functions related to hunting, defence and parasite deterrence. Toxins are produced either in situ by the toxic organism itself or associated symbionts, or acquired through diet. The ability to exploit toxins from external sources requires adaptations that prevent toxic effects on the consumer (autoresistance). Here, we examine genomic adaptations that could facilitate autoresistance to the diet-acquired potent neurotoxic alkaloid batrachotoxin (BTX) in New Guinean toxic birds. Our work documents two new toxic bird species and shows that toxic birds carry multiple mutations in the SCN4A gene that are under positive selection. This gene encodes the most common vertebrate muscle Nav channel (Nav1.4). Molecular docking results indicate that some of the mutations that are present in the pore-forming segment of the Nav channel, where BTX binds, could reduce its binding affinity. These mutations should therefore prevent the continuous opening of the sodium channels that BTX binding elicits, thereby preventing muscle paralysis and ultimately death. Although these mutations are different from those present in Neotropical Phyllobates poison dart frogs, they occur in the same segments of the Nav1.4 channel. Consequently, in addition to uncovering a greater diversity of toxic bird species than previously known, our work provides an intriguing example of molecular-level convergent adaptations allowing frogs and birds to ingest and use the same neurotoxin. This suggests that genetically modified Nav1.4 channels represent a key adaptation to BTX tolerance and exploitation across vertebrates.