Neuroimmunity: Physiology and Pathology.

Evolution has yielded multiple complex and complementary mechanisms to detect environmental danger and protect tissues from damage. The nervous system rapidly processes information and coordinates complex defense behaviors, and the immune system eliminates diverse threats by virtue of mobile, specialized cell populations. The two systems are tightly integrated, cooperating in local and systemic reflexes that restore homeostasis in response to tissue injury and infection. They further share a broad common language of cytokines, growth factors, and neuropeptides that enables bidirectional communication. However, this reciprocal cross talk permits amplification of maladaptive feedforward inflammatory loops that contribute to the development of allergy, autoimmunity, itch, and pain. Appreciating the immune and nervous systems as a holistic, coordinated defense system provides both new insights into inflammation and exciting opportunities for managing acute and chronic inflammatory diseases.

Nav1.8 in small dorsal root ganglion neurons contributes to vincristine-induced mechanical allodynia.

Vincristine-induced peripheral neuropathy is a common side effect of vincristine treatment, which is accompanied by pain and can be dose-limiting. The molecular mechanisms that underlie vincristine-induced pain are not well understood. We have established an animal model to investigate pathophysiological mechanisms of vincristine-induced pain. Our previous studies have shown that the tetrodotoxin-sensitive voltage-gated sodium channel Nav1.6 in medium-diameter dorsal root ganglion (DRG) neurons contributes to the maintenance of vincristine-induced allodynia. In this study, we investigated the effects of vincristine administration on excitability in small-diameter DRG neurons and whether the tetrodotoxin-resistant (TTX-R) Nav1.8 channels contribute to mechanical allodynia. Current-clamp recordings demonstrated that small DRG neurons become hyper-excitable following vincristine treatment, with both reduced current threshold and increased firing frequency. Using voltage-clamp recordings in small DRG neurons, we now show an increase in TTX-R current density and a -7.3 mV hyperpolarizing shift in the half-maximal potential (V1/2) of activation of Nav1.8 channels in vincristine-treated animals, which likely contributes to the hyperexcitability that we observed in these neurons. Notably, vincristine treatment did not enhance excitability of small DRG neurons from Nav1.8 knockout mice, and the development of mechanical allodynia was delayed but not abrogated in these mice. Together, our data suggest that sodium channel Nav1.8 in small DRG neurons contributes to the development of vincristine-induced mechanical allodynia.

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

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

Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider.

The King Baboon spider, Pelinobius muticus, is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus, but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including NaV1.8, KV2.1, and tetrodotoxin-sensitive NaV channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms.

Effects of inflammation on the properties of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative afferent mechanoreceptors in the hindpaw glabrous skin of mice.

We recently used Nav1.8-ChR2 mice in which Nav1.8-expressing afferents were optogenetically tagged to classify mechanosensitive afferents into Nav1.8-ChR2-positive and Nav1.8-ChR2-negative mechanoreceptors. We found that the former were mainly high threshold mechanoreceptors (HTMRs), while the latter were low threshold mechanoreceptors (LTMRs). In the present study, we further investigated whether the properties of these mechanoreceptors were altered following tissue inflammation. Nav1.8-ChR2 mice received a subcutaneous injection of saline or Complete Freund's Adjuvant (CFA) in the hindpaws. Using the hind paw glabrous skin-tibial nerve preparation and the pressure-clamped single-fiber recordings, we found that CFA-induced hind paw inflammation lowered the mechanical threshold of many Nav1.8-ChR2-positive Aß-fiber mechanoreceptors but heightened the mechanical threshold of many Nav1.8-ChR2-negative Aß-fiber mechanoreceptors. Spontaneous action potential impulses were not observed in Nav1.8-ChR2-positive Aß-fiber mechanoreceptors but occurred in Nav1.8-ChR2-negative Aß-fiber mechanoreceptors with a lower mechanical threshold in the saline goup, and a higher mechanical threshold in the CFA group. No significant change was observed in the mechanical sensitivity of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors and Nav1.8-ChR2-positive C-fiber mechanoreceptors following hind paw inflammation. Collectively, inflammation significantly altered the functional properties of both Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aß-fiber mechanoreceptors, which may contribute to mechanical allodynia during inflammation.

Unique electrophysiological property of a novel Nav1.7, Nav1.8, and Nav1.9 sodium channel blocker, ANP-230.

Voltage-gated sodium channel subtypes, Nav1.7, Nav1.8, and Nav1.9 are predominantly expressed in peripheral sensory neurons. Recent genetic studies have revealed that they are involved in pathological pain processing and that the blockade of Nav1.7, Nav1.8, or Nav1.9 will become a promising pharmacotherapy especially for neuropathic pain. A growing number of drug discovery programs have targeted either of the subtypes to obtain a selective inhibitor which can provide pain relief without affecting the cardiovascular and central nervous systems, though none of them has been approved yet. Here we describe the in vitro characteristics of ANP-230, a novel sodium channel blocker under clinical development. Surprisingly, ANP-230 was shown to block three pain-related subtypes, human Nav1.7, Nav1.8, and Nav1.9 with similar potency, but had only low inhibitory activity to human cardiac Nav1.5 channel and rat central Nav channels. The voltage clamp experiments using different step pulse protocols revealed that ANP-230 had a "tonic block" mode of action without state- and use-dependency. In addition, ANP-230 caused a depolarizing shift of the activation curve and decelerated gating kinetics in human Nav1.7-stably expressing cells. The depolarizing shift of activation curve was commonly observed in human Nav1.8-stably expressing cells as well as rat dorsal root ganglion neurons. These data suggested a quite unique mechanism of Nav channel inhibition by ANP-230. Finally, ANP-230 reduced excitability of rat dorsal root ganglion neurons in a concentration dependent manner. Collectively, these promising results indicate that ANP-230 could be a potent drug for neuropathic pain.

Identification of N-(((1S,3R,5S)-adamantan-1-yl)methyl)-3-((4-chlorophenyl)sulfonyl)benzenesulfonamide as novel Nav1.8 inhibitor with analgesic profile.

Chronic pain is a common and challenging clinical problem that significantly impacts patients' quality of life. The sodium channel Nav1.8 plays a crucial role in the occurrence and development of chronic pain, making it one of the key targets for treating chronic pain. In this article, we combined virtual screening with cell membrane chromatography techniques to establish a novel method for rapid high-throughput screening of selective Nav1.8 inhibitors. Using this approach, we identified a small molecule compound 6, which not only demonstrated high affinity and inhibitory activity against Nav1.8 but also exhibited significant inhibitory effects on CFA-induced chronic inflammatory pain. Compared to the positive drug VX-150, compound 6 showed a more prolonged analgesic effect, making it a promising candidate as a Nav1.8 inhibitor with potential clinical applications. This discovery provides a new therapeutic option for the treatment of chronic pain.

Frontiers in Acute Pain Management: Emerging Concepts in Pain Pathways and the Role of VX-548 as a Novel NaV1.8 Inhibitor: A Narrative Review.

PURPOSE OF REVIEW: Despite ongoing research into alternative postsurgical pain treatments, opioids remain widely used analgesics regardless of associated adverse effects, including dependence and overdose, as demonstrated throughout the current opioid crisis. This is likely related to a failure in proving the efficacy of alternative analgesics in clinical trials, despite strong evidence supporting the potential for effective analgesia through in vitro studies. While NaV1.7 and NaV1.8 channels have shown to be key components of pain perception, studies regarding pharmacological agents utilizing these channels as targets have largely failed to demonstrate the efficacy of these proposed analgesics when compared to current multimodal pain treatment regimens. RECENT FINDINGS: However, the novel NaV1.8 channel inhibitor, VX-548 has surpassed previously studied NaV1.8 inhibitors in clinical trials and continues to hold promise of a novel efficacious analgesic to potentially be utilized in multimodal pain treatment on postsurgical patients. Additionally, NaV1.8 is encoded by the SCN10A, which has been shown to be minimally expressed in the brain, suggesting a lower likelihood of adverse effects in the CNS, including dependence and abuse. Novel pharmacologic analgesics that are efficacious without the significant side effects associated with opioids have lacked meaningful development. However, recent clinical trials have shown promising results in the safety and efficacy of the pharmacological agent VX-548. Still, more clinical trials directly comparing the efficacy of VX-548 to standard of care post-surgical drugs, including opioids like morphine and hydromorphone are needed to demonstrate the long-term viability of the agent replacing current opioids with an unfavorable side effect profile.

NaV1.8 as Proarrhythmic Target in a Ventricular Cardiac Stem Cell Model.

The sodium channel NaV1.8, encoded by the SCN10A gene, has recently emerged as a potential regulator of cardiac electrophysiology. We have previously shown that NaV1.8 contributes to arrhythmogenesis by inducing a persistent Na+ current (late Na+ current, INaL) in human atrial and ventricular cardiomyocytes (CM). We now aim to further investigate the contribution of NaV1.8 to human ventricular arrhythmogenesis at the CM-specific level using pharmacological inhibition as well as a genetic knockout (KO) of SCN10A in induced pluripotent stem cell CM (iPSC-CM). In functional voltage-clamp experiments, we demonstrate that INaL was significantly reduced in ventricular SCN10A-KO iPSC-CM and in control CM after a specific pharmacological inhibition of NaV1.8. In contrast, we did not find any effects on ventricular APD90. The frequency of spontaneous sarcoplasmic reticulum Ca2+ sparks and waves were reduced in SCN10A-KO iPSC-CM and control cells following the pharmacological inhibition of NaV1.8. We further analyzed potential triggers of arrhythmias and found reduced delayed afterdepolarizations (DAD) in SCN10A-KO iPSC-CM and after the specific inhibition of NaV1.8 in control cells. In conclusion, we show that NaV1.8-induced INaL primarily impacts arrhythmogenesis at a subcellular level, with minimal effects on systolic cellular Ca2+ release. The inhibition or knockout of NaV1.8 diminishes proarrhythmic triggers in ventricular CM. In conjunction with our previously published results, this work confirms NaV1.8 as a proarrhythmic target that may be useful in an anti-arrhythmic therapeutic strategy.

Peripheral temperature dysregulation associated with functionally altered NaV1.8 channels.

The voltage-gated sodium channel NaV1.8 is prominently expressed in the soma and axons of small-caliber sensory neurons, and pathogenic variants of the corresponding gene SCN10A are associated with peripheral pain and autonomic dysfunction. While most disease-associated SCN10A variants confer gain-of-function properties to NaV1.8, resulting in hyperexcitability of sensory neurons, a few affect afferent excitability through a loss-of-function mechanism. Using whole-exome sequencing, we here identify a rare heterozygous SCN10A missense variant resulting in alteration p.V1287I in NaV1.8 in a patient with a 15-year history of progressively worsening temperature dysregulation in the distal extremities, particularly in the feet. Further symptoms include increasingly intensifying tingling and numbness in the fingers and increased sweating. To assess the impact of p.V1287I on channel function, we performed voltage-clamp recordings demonstrating that the alteration confers loss- and gain-of-function characteristics to NaV1.8 characterized by a right-shifted voltage dependence of channel activation and inactivation. Current-clamp recordings from transfected mouse dorsal root ganglion neurons further revealed that NaV1.8-V1287I channels broaden the action potentials of sensory neurons and increase their firing rates in response to depolarizing current stimulations, indicating a gain-of-function mechanism of the variant at the cellular level in a heterozygous setting. The data support the hypothesis that the properties of NaV1.8 p.V1287I are causative for the patient's symptoms and that nonpainful peripheral paresthesias should be considered part of the clinical spectrum of NaV1.8-associated disorders.

Trigeminal neurons control immune-bone cell interaction and metabolism in apical periodontitis.

Apical periodontitis (AP) is an inflammatory disease occurring following tooth infection with distinct osteolytic activity. Despite increasing evidence that sensory neurons participate in regulation of non-neuronal cells, their role in the development of AP is largely unknown. We hypothesized that trigeminal ganglia (TG) Nav1.8+ nociceptors regulate bone metabolism changes in response to AP. A selective ablation of nociceptive neurons in Nav1.8Cre/Diphtheria toxin A (DTA)Lox mouse line was used to evaluate the development and progression of AP using murine model of infection-induced AP. Ablation of Nav1.8+ nociceptors had earlier progression of AP with larger osteolytic lesions. Immunohistochemical and RNAscope analyses demonstrated greater number of macrophages, T-cells, osteoclast and osteoblast precursors and an increased RANKL:OPG ratio at earlier time points among Nav1.8Cre/ DTALox mice. There was an increased expression of IL-1α and IL-6 within lesions of nociceptor-ablated mice. Further, co-culture experiments demonstrated that TG neurons promoted osteoblast mineralization and inhibited osteoclastic function. The findings suggest that TG Nav1.8+ neurons contribute to modulation of the AP development by delaying the influx of immune cells, promoting osteoblastic differentiation, and decreasing osteoclastic activities. This newly uncovered mechanism could become a therapeutic strategy for the treatment of AP and minimize the persistence of osteolytic lesions in refractory cases.

Natural Compounds Purified from the Leaves of Aristotelia chilensis: Makomakinol, a New Alkaloid and the Effect of Aristoteline and Hobartine on NaV Channels.

Aristotelia chilensis or "maqui" is a tree native to Chile used in the folk medicine of the Mapuche people as an anti-inflammatory agent for the treatment of digestive ailments, fever, and skin lesions. Maqui fruits are black berries which are considered a "superfruit" with notable potential health benefits, promoted to be an antioxidant, cardioprotective, and anti-inflammatory. Maqui leaves contain non-iridoid monoterpene indole alkaloids which have previously been shown to act on nicotinic acetylcholine receptors, potassium channels, and calcium channels. Here, we isolated a new alkaloid from maqui leaves, now called makomakinol, together with the known alkaloids aristoteline, hobartine, and 3-formylindole. Moreover, the polyphenols quercetine, ethyl caffeate, and the terpenes, dihydro-ß-ionone and terpin hydrate, were also obtained. In light of the reported analgesic and anti-nociceptive properties of A. chilensis, in particular a crude mixture of alkaloids containing aristoteline and hobartinol (PMID 21585384), we therefore evaluated the activity of aristoteline and hobartine on NaV1.8, a key NaV isoform involved in nociception, using automated whole-cell patch-clamp electrophysiology. Aristoteline and hobartine both inhibited Nav1.8 with an IC50 of 68 ± 3 µM and 54 ± 1 µM, respectively. Hobartine caused a hyperpolarizing shift of the voltage-dependence of the activation, whereas aristoteline did not change the voltage-dependence of the activation or inactivation. The inhibitory activity of these alkaloids on NaV channels may contribute to the reported analgesic properties of Aristotelia chilensis used by the Mapuche people.

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.

Cannabidiol Inhibition of Murine Primary Nociceptors: Tight Binding to Slow Inactivated States of Nav1.8 Channels.

The nonpsychoactive phytocannabinoid cannabidiol (CBD) has been shown to have analgesic effects in animal studies but little is known about its mechanism of action. We examined the effects of CBD on intrinsic excitability of primary pain-sensing neurons. Studying acutely dissociated capsaicin-sensitive mouse DRG neurons at 37°C, we found that CBD effectively inhibited repetitive action potential firing, from 15-20 action potentials evoked by 1 s current injections in control to 1-3 action potentials with 2 µm CBD. Reduction of repetitive firing was accompanied by a reduction of action potential height, widening of action potentials, reduction of the afterhyperpolarization, and increased propensity to enter depolarization block. Voltage-clamp experiments showed that CBD inhibited both TTX-sensitive and TTX-resistant (TTX-R) sodium currents in a use-dependent manner. CBD showed strong state-dependent inhibition of TTX-R channels, with fast binding to inactivated channels during depolarizations and slow unbinding on repolarization. CBD alteration of channel availability at various voltages suggested that CBD binds especially tightly [Kd (dissociation constant), ∼150 nm] to the slow inactivated state of TTX-R channels, which can be substantially occupied at voltages as negative as -40 mV. Remarkably, CBD was more potent in inhibiting TTX-R channels and inhibiting action potential firing than the local anesthetic bupivacaine. We conclude that CBD might produce some of its analgesic effects by direct effects on neuronal excitability, with tight binding to the slow inactivated state of Nav1.8 channels contributing to effective inhibition of repetitive firing by modest depolarizations.SIGNIFICANCE STATEMENT Cannabidiol (CBD) has been shown to inhibit pain in various rodent models, but the mechanism of this effect is unknown. We describe the ability of CBD to inhibit repetitive action potential firing in primary nociceptive neurons from mouse dorsal root ganglia and analyze the effects on voltage-dependent sodium channels. We find that CBD interacts with TTX-resistant sodium channels in a state-dependent manner suggesting particularly tight binding to slow inactivated states of Nav1.8 channels, which dominate the overall inactivation of Nav1.8 channels for small maintained depolarizations from the resting potential. The results suggest that CBD can exert analgesic effects in part by directly inhibiting repetitive firing of primary nociceptors and suggest a strategy of identifying compounds that bind selectively to slow inactivated states of Nav1.8 channels for developing effective analgesics.

New approaches to the design of analgesic medicinal substances.

A gamma-pyrone derivative, comenic acid, activates the opioid-like receptor-mediated signaling pathway that modulates the NaV1.8 channels in the primary sensory neuron membrane. These channels are responsible for the generation of the nociceptive signal; therefore, gamma-pyrones have great therapeutic potential as analgesics, and this effect deserves a deeper understanding. The novelty of our approach to the design of a medicinal substance is based on a combination of the data obtained from living neurons using very sensitive physiological methods and the results of quantum chemical calculations. This approach allows the correlation of the molecular structure of gamma-pyrones with their ability to evoke a physiological response of the neuron. Comenic acid can bind to two calcium cations. One of them is chelated by the carbonyl and hydroxyl functional groups, while the other forms a salt bond with the carboxylate anion. Calcium-bound gamma-pyrones have fundamentally different electrostatic properties from free gamma-pyrone molecules. These two calcium ions are key elements involved in ligand-receptor binding. It is very likely that ion-ionic interactions between these cations and anionic functional groups of the opioid-like receptor activate the latter. The calculated intercationic distance of 9.5 Å is a structural criterion for effective ligand-receptor binding of calcium-bound gamma-pyrones.

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.

Cold sensing by NaV1.8-positive and NaV1.8-negative sensory neurons.

The ability to detect environmental cold serves as an important survival tool. The sodium channels NaV1.8 and NaV1.9, as well as the TRP channel Trpm8, have been shown to contribute to cold sensation in mice. Surprisingly, transcriptional profiling shows that NaV1.8/NaV1.9 and Trpm8 are expressed in nonoverlapping neuronal populations. Here we have used in vivo GCaMP3 imaging to identify cold-sensing populations of sensory neurons in live mice. We find that ∼80% of neurons responsive to cold down to 1 °C do not express NaV1.8, and that the genetic deletion of NaV1.8 does not affect the relative number, distribution, or maximal response of cold-sensitive neurons. Furthermore, the deletion of NaV1.8 had no observable effect on transient cold-induced (≥5 °C) behaviors in mice, as measured by the cold-plantar, cold-plate (5 and 10 °C), or acetone tests. In contrast, nocifensive-like behavior to extreme cold-plate stimulation (-5 °C) was completely absent in mice lacking NaV1.8. Fluorescence-activated cell sorting (FACS) and subsequent microarray analysis of sensory neurons activated at 4 °C identified an enriched repertoire of ion channels, which include the Trp channel Trpm8 and potassium channel Kcnk9, that are potentially required for cold sensing above freezing temperatures in mouse DRG neurons. These data demonstrate the complexity of cold-sensing mechanisms in mouse sensory neurons, revealing a principal role for NaV1.8-negative neurons in sensing both innocuous and acute noxious cooling down to 1 °C, while NaV1.8-positive neurons are likely responsible for the transduction of prolonged extreme cold temperatures, where tissue damage causes pan-nociceptor activation.

Arginine-Containing Tripeptides as Analgesic Substances: The Possible Mechanism of Ligand-Receptor Binding to the Slow Sodium Channel.

Two short arginine-containing tripeptides, H-Arg-Arg-Arg-OH (TP1) and Ac-Arg-Arg-Arg-NH2 (TP2), have been shown by the patch-clamp method to modulate the NaV1.8 channels of DRG primary sensory neurons, which are responsible for the generation of nociceptive signals. Conformational analysis of the tripeptides indicates that the key role in the ligand-receptor binding of TP1 and TP2 to the NaV1.8 channel is played by two positively charged guanidinium groups of the arginine side chains located at the characteristic distance of ~9 Å from each other. The tripeptide effect on the NaV1.8 channel activation gating device has been retained when the N- and C-terminal groups of TP1 were structurally modified to TP2 to protect the attacking peptide from proteolytic cleavage by exopeptidases during its delivery to the molecular target, the NaV1.8 channel. As demonstrated by the organotypic tissue culture method, the agents do not affect the DRG neurite growth, which makes it possible to expect the absence of adverse side effects at the tissue level upon administration of TP1 and TP2. The data obtained indicate that both tripeptides can have great therapeutic potential as novel analgesic medicinal substances.

Role of the Guanidinium Groups in Ligand-Receptor Binding of Arginine-Containing Short Peptides to the Slow Sodium Channel: Quantitative Approach to Drug Design of Peptide Analgesics.

Several arginine-containing short peptides have been shown by the patch-clamp method to effectively modulate the NaV1.8 channel activation gating system, which makes them promising candidates for the role of a novel analgesic medicinal substance. As demonstrated by the organotypic tissue culture method, all active and inactive peptides studied do not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects on the tissue level upon their medicinal administration. The conformational analysis of Ac-RAR-NH2, Ac-RER-NH2, Ac-RAAR-NH2, Ac-REAR-NH2, Ac-RERR-NH2, Ac-REAAR-NH2, Ac-PRERRA-NH2, and Ac-PRARRA-NH2 has made it possible to find the structural parameter, the value of which is correlated with the target physiological effect of arginine-containing short peptides. The distances between the positively charged guanidinium groups of the arginine side chains involved in intermolecular ligand-receptor ion-ion bonds between the attacking peptide molecules and the NaV1.8 channel molecule should fall within a certain range, the lower threshold of which is estimated to be around 9 Å. The distance values have been calculated to be below 9 Å in the inactive peptide molecules, except for Ac-RER-NH2, and in the range of 9-12 Å in the active peptide molecules.

Scorpion Neurotoxin Syb-prII-1 Exerts Analgesic Effect through Nav1.8 Channel and MAPKs Pathway.

Trigeminal neuralgia (TN) is a common type of peripheral neuralgia in clinical practice, which is usually difficult to cure. Common analgesic drugs are difficult for achieving the desired analgesic effect. Syb-prII-1 is a ß-type scorpion neurotoxin isolated from the scorpion venom of Buthus martensi Karsch (BmK). It has an important influence on the voltage-gated sodium channel (VGSCs), especially closely related to Nav1.8 and Nav1.9. To explore whether Syb-prII-1 has a good analgesic effect on TN, we established the Sprague Dawley (SD) rats' chronic constriction injury of the infraorbital nerve (IoN-CCI) model. Behavioral, electrophysiological, Western blot, and other methods were used to verify the model. It was found that Syb-prII-1 could significantly relieve the pain behavior of IoN-CCI rats. After Syb-prII-1 was given, the phosphorylation level of the mitogen-activated protein kinases (MAPKs) pathway showed a dose-dependent decrease after IoN-CCI injury. Moreover, Syb-prII-1(4.0 mg/kg) could significantly change the steady-state activation and inactivation curves of Nav1.8. The steady-state activation and inactivation curves of Nav1.9 were similar to those of Nav1.8, but there was no significant difference. It was speculated that it might play an auxiliary role. The binding mode, critical residues, and specific interaction type of Syb-prII-1 and VSD2rNav1.8 were clarified with computational simulation methods. Our results indicated that Syb-prII-1 could provide a potential treatment for TN by acting on the Nav1.8 target.