Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin.

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.

Nav1.7 as a chondrocyte regulator and therapeutic target for osteoarthritis.

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2 and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief.

Voltage-gated sodium (NaV) channels control the upstroke of the action potentials in excitable cells. Multiple studies have shown distinct roles of NaV channel subtypes in human physiology and diseases, but subtype-specific therapeutics are lacking and the current efforts have been limited to small molecules. Here, we present a monoclonal antibody that targets the voltage-sensor paddle of NaV1.7, the subtype critical for pain sensation. This antibody not only inhibits NaV1.7 with high selectivity, but also effectively suppresses inflammatory and neuropathic pain in mice. Interestingly, the antibody inhibits acute and chronic itch despite well-documented differences in pain and itch modulation. Using this antibody, we discovered that NaV1.7 plays a key role in spinal cord nociceptive and pruriceptive synaptic transmission. Our studies reveal that NaV1.7 is a target for itch management, and the antibody has therapeutic potential for suppressing pain and itch. Our antibody strategy may have broad applications for voltage-gated cation channels.

Conotoxins Targeting Voltage-Gated Sodium Ion Channels.

Voltage-gated sodium (NaV) channels are intimately involved in the generation and transmission of action potentials, and dysfunction of these channels may contribute to nervous system diseases, such as epilepsy, neuropathic pain, psychosis, autism, and cardiac arrhythmia. Many venom peptides selectively act on NaV channels. These include conotoxins, which are neurotoxins secreted by cone snails for prey capture or self-defense but which are also valuable pharmacological tools for the identification and/or treatment of human diseases. Typically, conotoxins contain two or three disulfide bonds, and these internal crossbraces contribute to conotoxins having compact, well defined structures and high stability. Of the conotoxins containing three disulfide bonds, some selectively target mammalian NaV channels and can block, stimulate, or modulate these channels. Such conotoxins have great potential to serve as pharmacological tools for studying the functions and characteristics of NaV channels or as drug leads for neurologic diseases related to NaV channels. Accordingly, discovering or designing conotoxins targeting NaV channels with high potency and selectivity is important. The amino acid sequences, disulfide bond connectivity, and three-dimensional structures are key factors that affect the biological activity of conotoxins, and targeted synthetic modifications of conotoxins can greatly improve their activity and selectivity. This review examines NaV channel-targeted conotoxins, focusing on their structures, activities, and designed modifications, with a view toward expanding their applications. SIGNIFICANCE STATEMENT: NaV channels are crucial in various neurologic diseases. Some conotoxins selectively target NaV channels, causing either blockade or activation, thus enabling their use as pharmacological tools for studying the channels' characteristics and functions. Conotoxins also have promising potential to be developed as drug leads. The disulfide bonds in these peptides are important for stabilizing their structures, thus leading to enhanced specificity and potency. Together, conotoxins targeting NaV channels have both immediate research value and promising future application prospects.

The Role of Voltage-Gated Sodium Channels in Pain Signaling.

Acute pain signaling has a key protective role and is highly evolutionarily conserved. Chronic pain, however, is maladaptive, occurring as a consequence of injury and disease, and is associated with sensitization of the somatosensory nervous system. Primary sensory neurons are involved in both of these processes, and the recent advances in understanding sensory transduction and human genetics are the focus of this review. Voltage-gated sodium channels (VGSCs) are important determinants of sensory neuron excitability: they are essential for the initial transduction of sensory stimuli, the electrogenesis of the action potential, and neurotransmitter release from sensory neuron terminals. Nav1.1, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 are all expressed by adult sensory neurons. The biophysical characteristics of these channels, as well as their unique expression patterns within subtypes of sensory neurons, define their functional role in pain signaling. Changes in the expression of VGSCs, as well as posttranslational modifications, contribute to the sensitization of sensory neurons in chronic pain states. Furthermore, gene variants in Nav1.7, Nav1.8, and Nav1.9 have now been linked to human Mendelian pain disorders and more recently to common pain disorders such as small-fiber neuropathy. Chronic pain affects one in five of the general population. Given the poor efficacy of current analgesics, the selective expression of particular VGSCs in sensory neurons makes these attractive targets for drug discovery. The increasing availability of gene sequencing, combined with structural modeling and electrophysiological analysis of gene variants, also provides the opportunity to better target existing therapies in a personalized manner.

Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

Exenatide reduces atrial fibrillation susceptibility by inhibiting hKv1.5 and hNav1.5 channels.

Exenatide, a promising cardioprotective agent, protects against cardiac structural remodeling and diastolic dysfunction. Combined blockade of sodium and potassium channels is valuable for managing atrial fibrillation (AF). Here, we explored whether exenatide displayed anti-AF effects by inhibiting human Kv1.5 and Nav1.5 channels. We used the whole-cell patch-clamp technique to investigate the effects of exenatide on hKv1.5 and hNav1.5 channels expressed in human embryonic kidney 293 cells and studied the effects of exenatide on action potential (AP) and other cardiac ionic currents in rat atrial myocytes. Additionally, an electrical mapping system was used to explore the effects of exenatide on electrical properties and AF activity in isolated rat hearts. Finally, a rat AF model, established using acetylcholine and calcium chloride, was employed to evaluate the anti-AF potential of exenatide in rats. Exenatide reversibly suppressed IKv1.5 with IC50 of 3.08 µM, preferentially blocked the hKv1.5 channel in its closed state, and positively shifted the voltage-dependent activation curve. Exenatide also reversibly inhibited INav1.5 with IC50 of 3.30 µM, negatively shifted the voltage-dependent inactivation curve, and slowed its recovery from inactivation with significant use-dependency at 5 and 10 Hz. Furthermore, exenatide prolonged AP duration and suppressed the sustained K+ current (Iss) and transient outward K+ current (Ito), but without inhibition of L-type Ca2+ current (ICa,L) in rat atrial myocytes. Exenatide prevented AF incidence and duration in rat hearts and rats. These findings demonstrate that exenatide inhibits IKv1.5 and INav1.5in vitro and reduces AF susceptibility in isolated rat hearts and rats.

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.

Design, synthesis, and mechanism of action of novel µ-conotoxin KIIIA analogues for inhibition of the voltage-gated sodium channel Nav1.7.

µ-Conotoxin KIIIA, a selective blocker of sodium channels, has strong inhibitory activity against several Nav isoforms, including Nav1.7, and has potent analgesic effects, but it contains three pairs of disulfide bonds, making structural modification difficult and synthesis complex. To circumvent these difficulties, we designed and synthesized three KIIIA analogues with one disulfide bond deleted. The most active analogue, KIIIA-1, was further analyzed, and its binding pattern to hNav1.7 was determined by molecular dynamics simulations. Guided by the molecular dynamics computational model, we designed and tested 32 second-generation and 6 third-generation analogues of KIIIA-1 on hNav1.7 expressed in HEK293 cells. Several analogues showed significantly improved inhibitory activity on hNav1.7, and the most potent peptide, 37, was approximately 4-fold more potent than the KIIIA Isomer I and 8-fold more potent than the wildtype (WT) KIIIA in inhibiting hNav1.7 current. Intraperitoneally injected 37 exhibited potent in vivo analgesic activity in a formalin-induced inflammatory pain model, with activity reaching ∼350-fold of the positive control drug morphine. Overall, peptide 37 has a simplified disulfide-bond framework and exhibits potent in vivo analgesic effects and has promising potential for development as a pain therapy in the future.

Ranolazine: a potential anti-metastatic drug targeting voltage-gated sodium channels.

BACKGROUND: Multi-faceted evidence from a range of cancers suggests strongly that de novo expression of voltage-gated sodium channels (VGSCs) plays a significant role in driving cancer cell invasiveness. Under hypoxic conditions, common to growing tumours, VGSCs develop a persistent current (INaP) which can be blocked selectively by ranolazine. METHODS: Several different carcinomas were examined. We used data from a range of experimental approaches relating to cellular invasiveness and metastasis. These were supplemented by survival data mined from cancer patients. RESULTS: In vitro, ranolazine inhibited invasiveness of cancer cells especially under hypoxia. In vivo, ranolazine suppressed the metastatic abilities of breast and prostate cancers and melanoma. These data were supported by a major retrospective epidemiological study on breast, colon and prostate cancer patients. This showed that risk of dying from cancer was reduced by ca.60% among those taking ranolazine, even if this started 4 years after the diagnosis. Ranolazine was also shown to reduce the adverse effects of chemotherapy on heart and brain. Furthermore, its anti-cancer effectiveness could be boosted by co-administration with other drugs. CONCLUSIONS: Ranolazine, alone or in combination with appropriate therapies, could be reformulated as a safe anti-metastatic drug offering many potential advantages over current systemic treatment modalities.

Inhibiting Nav1.7 channels in pulpitis: An in vivo study on neuronal hyperexcitability.

Pulpitis constitutes a significant challenge in clinical management due to its impact on peripheral nerve tissue and the persistence of chronic pain. Despite its clinical importance, the correlation between neuronal activity and the expression of voltage-gated sodium channel 1.7 (Nav1.7) in the trigeminal ganglion (TG) during pulpitis is less investigated. The aim of this study was to examine the relationship between experimentally induced pulpitis and Nav1.7 expression in the TG and to investigate the potential of selective Nav1.7 modulation to attenuate TG abnormal activity associated with pulpitis. Acute pulpitis was induced at the maxillary molar (M1) using allyl isothiocyanate (AITC). The mice were divided into three groups: control, pulpitis model, and pulpitis model treated with ProTx-II, a selective Nav1.7 channel inhibitor. After three days following the surgery, we conducted a recording and comparative analysis of the neural activity of the TG utilizing in vivo optical imaging. Then immunohistochemistry and Western blot were performed to assess changes in the expression levels of extracellular signal-regulated kinase (ERK), c-Fos, collapsin response mediator protein-2 (CRMP2), and Nav1.7 channels. The optical imaging result showed significant neurological excitation in pulpitis TGs. Nav1.7 expressions exhibited upregulation, accompanied by signaling molecular changes suggestive of inflammation and neuroplasticity. In addition, inhibition of Nav1.7 led to reduced neural activity and subsequent decreases in ERK, c-Fos, and CRMP2 levels. These findings suggest the potential for targeting overexpressed Nav1.7 channels to alleviate pain associated with pulpitis, providing practical pain management strategies.

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.

The Efficacy of I Na Block to Cardiovert Atrial Fibrillation Is Enhanced by Inhibition of I K1.

ABSTRACT: There is a need for more efficient pharmacological cardioversion of atrial fibrillation (AF). We tested the hypothesis that inhibition of I K1 significantly enhances the efficacy of I Na block to depress atrial excitability and to cardiovert AF. The study was conducted in canine isolated arterially perfused right atrial preparations with rim of ventricular tissue. AF was induced in the presence of acetylcholine (ACh; 0.5 µM). BaCl 2 (10 µM) was used to inhibit I K1 and flecainide (1.5 µM) to block I Na . Sustained AF (>45 minutes) was recorded in 100% atria (5/5) in the presence of ACh alone. Flecainide cardioverted AF in 50% of atria (4/8), BaCl 2 in 0% (0/5), and their combination in 100% (5/5). AF cardioversion occurred in 15 ± 9 minutes with flecainide alone (n = 4) and in 8 ± 9 minutes with the combination (n = 5). Following drug-induced AF cardioversion, AF was inducible in 4/4 atria with flecainide alone (≤5 minutes duration) and in 2/5 atria with the combination (≤30 seconds duration). Atrial excitability was significantly more depressed by combined versus monotherapies. There was little to no effect on ventricular excitability under any condition tested. Thus, inhibition of I K1 significantly enhances the efficacy of flecainide to depress atrial excitability and to cardiovert AF in our experimental setting. A combination of I Na and I K1 inhibition may be an effective approach for cardioversion of AF.

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.

Blocking NaV1.8 regulates atrial fibrillation inducibility and cardiac conduction after myocardial infarction.

BACKGROUND: The role of NaV1.8 impacts in atrial fibrillation susceptibility after myocardial infarction remains only partially understood. We studied the effect of blocking NaV1.8 in the cardiac ganglionated plexi (GP) on the atrial fibrillation inducibility and cardiac conduction in the myocardial infarction model. METHODS: Eighteen male beagles were randomly enrolled. Left anterior descending coronary artery was ligated to created myocardial infarction model. Four weeks after surgery, NaV1.8 blocker A-803,467 (n = 9) or DMSO (n = 9, control) was injected into the four cardiac major GPs. Sinus rate, ventricular rate during atrial fibrillation, PR interval, atrial effective refractory period, atrial fibrillation duration and the cumulative window of atrial vulnerability were measured before and 60 min after A-803,467 injection. RESULTS: Administration of A-803,467 significantly increased sinus rate, shortened PR interval and increased ventricular rate during atrial fibrillation compared to control. A-803,467 also significantly shortened atrial effective refractory period, prolonged atrial fibrillation duration and increased the cumulative window of atrial vulnerability. A-803,467 suppressed the slowing of heart rate response to high-frequency electrical stimulation of the anterior right GP, which was used as the surrogate marker for GP function. Double staining of ChAT and NaV1.8 demonstrated colocalization of ChAT and NaV1.8 in canine GPs. CONCLUSIONS: Blocking NaV1.8 in the cardiac GP may modulate atrial fibrillation inducibility and cardiac conduction after myocardial infarction, and the underlying mechanism may be associated with the regulation of the neural activity of the cardiac GP.

Recent advances in small molecule Nav 1.7 inhibitors for cancer pain management.

The dorsal root ganglion (DRG) is the primary neuron responsible for transmitting peripheral pain signals to the central nervous system and plays a crucial role in pain transduction. Modulation of DRG excitability is considered a viable approach for pain management. Neuronal excitability is intricately linked to the ion channels on the neurons. The small and medium-sized DRG neurons are chiefly engaged in pain conduction and have high levels of TTX-S sodium channels, with Nav1.7 accounting for approximately 80% of the current. Voltage-gated sodium channel (VGSC or Nav) blockers are vital targets for the management of central nervous system diseases, particularly chronic pain. VGSCs play a key role in controlling cellular excitability. Clinical research has shown that Nav1.7 plays a crucial role in pain sensation, and there is strong genetic evidence linking Nav1.7 and its encoding gene SCN9A gene to painful disorders in humans. Many studies have shown that Nav1.7 plays an important role in pain management. The role of Nav1.7 in pain signaling pathways makes it an attractive target for the potential development of new pain drugs. Meanwhile, understanding the architecture of Nav1.7 may help to develop the next generation of painkillers. This review provides updates on the recently reported molecular inhibitors targeting the Nav1.7 pathway, summarizes their structure-activity relationships (SARs), and discusses their therapeutic effects on painful diseases. Pharmaceutical chemists are working to improve the therapeutic index of Nav1.7 inhibitors, achieve better analgesic effects, and reduce side effects. We hope that this review will contribute to the development of novel Nav1.7 inhibitors as potential drugs.

Effectiveness and safety of mexiletine versus placebo in patients with myotonia: a systematic review and meta-analysis.

BACKGROUND: The rare nature of dystrophic and non-dystrophic myotonia has limited the available evidence on the efficacy of mexiletine as a potential treatment. To address this gap, we conducted a systematic review and meta-analysis to evaluate the effectiveness and safety of mexiletine for both dystrophic and non-dystrophic myotonic patients. METHODS: The search was conducted on various electronic databases up to March 2023, for randomized clinical trials (RCTs) comparing mexiletine versus placebo in myotonic patients. A risk of bias assessment was carried out, and relevant data was extracted manually into an online sheet. RevMan software (version 5.4) was employed for analysis. RESULTS: A total of five studies, comprising 186 patients, were included in the meta-analysis. Our findings showed that mexiletine was significantly more effective than placebo in improving stiffness score (SMD = - 1.19, 95% CI [- 1.53, - 0.85]), as well as in reducing hand grip myotonia (MD = - 1.36 s, 95% CI [- 1.83, - 0.89]). Mexiletine also significantly improved SF-36 Physical and Mental Component Score in patients with non-dystrophic myotonia only. Regarding safety, mexiletine did not significantly alter ECG parameters but was associated with greater gastrointestinal symptoms (GIT) compared to placebo (RR 3.7, 95% CI [1.79, 7.64]). Other adverse events showed no significant differences. CONCLUSION: The results support that mexiletine is effective and safe in myotonic patients; however, it is associated with a higher risk of GIT symptoms. Due to the scarcity of published RCTs and the prevalence of GIT symptoms, we recommend further well-designed RCTs testing various drug combinations to reduce GIT symptoms.

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.

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.