Human Voltage-gated Sodium Channel Research Articles

The road to evolution of ProTx2: how to be a subtype-specific inhibition of human Nav1.7.

The human voltage-gated sodium channel Nav1.7 is a widely proven target for analgesic drug studies. ProTx2, a 30-residue polypeptide from Peruvian green tarantula venom, shows high specificity to activity against human Nav1.7, suggesting its potential to become a non-addictive analgesic. However, its high sensitivity to human Nav1.4 raises concerns about muscle side effects. Here, we engineered three mutants (R13A, R13D, and K27Y) of ProTx2 to evaluate their pharmacological activities toward Nav1.7 and Nav1.4. It is demonstrated that the mutant R13D maintained the analgesic effect in mice while dramatically reducing its muscle toxicity compared with ProTx2. The main reason is the formation of a strong electrostatic interaction between R13D and the negatively charged amino acid residues in DII/S3-S4 of Nav1.7, which is absent in Nav1.4. This study advances our understanding and insights on peptide toxins, paving the way for safer, effective non-addictive analgesic development.

Structural modeling of ion channels using AlphaFold2, RoseTTAFold2, and ESMFold

ABSTRACT Ion channels play key roles in human physiology and are important targets in drug discovery. The atomic-scale structures of ion channels provide invaluable insights into a fundamental understanding of the molecular mechanisms of channel gating and modulation. Recent breakthroughs in deep learning-based computational methods, such as AlphaFold, RoseTTAFold, and ESMFold have transformed research in protein structure prediction and design. We review the application of AlphaFold, RoseTTAFold, and ESMFold to structural modeling of ion channels using representative voltage-gated ion channels, including human voltage-gated sodium (NaV) channel - NaV1.8, human voltage-gated calcium (CaV) channel – CaV1.1, and human voltage-gated potassium (KV) channel – KV1.3. We compared AlphaFold, RoseTTAFold, and ESMFold structural models of NaV1.8, CaV1.1, and KV1.3 with corresponding cryo-EM structures to assess details of their similarities and differences. Our findings shed light on the strengths and limitations of the current state-of-the-art deep learning-based computational methods for modeling ion channel structures, offering valuable insights to guide their future applications for ion channel research.

Elucidating molecular mechanisms of protoxin-II state-specific binding to the human NaV1.7 channel.

Human voltage-gated sodium (hNaV) channels are responsible for initiating and propagating action potentials in excitable cells, and mutations have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels are expressed in peripheral neurons and are promising targets for pain therapy. The tarantula venom peptide protoxin-II (PTx2) has high selectivity for hNaV1.7 and is a valuable scaffold for designing novel therapeutics to treat pain. Here, we used computational modeling to study the molecular mechanisms of the state-dependent binding of PTx2 to hNaV1.7 voltage-sensing domains (VSDs). Using Rosetta structural modeling methods, we constructed atomistic models of the hNaV1.7 VSD II and IV in the activated and deactivated states with docked PTx2. We then performed microsecond-long all-atom molecular dynamics (MD) simulations of the systems in hydrated lipid bilayers. Our simulations revealed that PTx2 binds most favorably to the deactivated VSD II and activated VSD IV. These state-specific interactions are mediated primarily by PTx2's residues R22, K26, K27, K28, and W30 with VSD and the surrounding membrane lipids. Our work revealed important protein-protein and protein-lipid contacts that contribute to high-affinity state-dependent toxin interaction with the channel. The workflow presented will prove useful for designing novel peptides with improved selectivity and potency for more effective and safe treatment of pain.

Structure-function and rational design of a spider toxin Ssp1a at human voltage-gated sodium channel subtypes.

The structure-function and optimization studies of NaV-inhibiting spider toxins have focused on developing selective inhibitors for peripheral pain-sensing NaV1.7. With several NaV subtypes emerging as potential therapeutic targets, structure-function analysis of NaV-inhibiting spider toxins at such subtypes is warranted. Using the recently discovered spider toxin Ssp1a, this study extends the structure-function relationships of NaV-inhibiting spider toxins beyond NaV1.7 to include the epilepsy target NaV1.2 and the pain target NaV1.3. Based on these results and docking studies, we designed analogues for improved potency and/or subtype-selectivity, with S7R-E18K-rSsp1a and N14D-P27R-rSsp1a identified as promising leads. S7R-E18K-rSsp1a increased the rSsp1a potency at these three NaV subtypes, especially at NaV1.3 (∼10-fold), while N14D-P27R-rSsp1a enhanced NaV1.2/1.7 selectivity over NaV1.3. This study highlights the challenge of developing subtype-selective spider toxin inhibitors across multiple NaV subtypes that might offer a more effective therapeutic approach. The findings of this study provide a basis for further rational design of Ssp1a and related NaSpTx1 homologs targeting NaV1.2, NaV1.3 and/or NaV1.7 as research tools and therapeutic leads.

Molecular Dynamics Simulations of Ion Permeation in Human Voltage-Gated Sodium Channels.

The recent determination of cryo-EM structures of voltage-gated sodium (Nav) channels has revealed many details of these proteins. However, knowledge of ionic permeation through the Nav pore remains limited. In this work, we performed atomistic molecular dynamics (MD) simulations to study the structural features of various neuronal Nav channels based on homology modeling of the cryo-EM structure of the human Nav1.4 channel and, in addition, on the recently resolved configuration for Nav1.2. In particular, single Na+ permeation events during standard MD runs suggest that the ion resides in the inner part of the Nav selectivity filter (SF). On-the-fly free energy parametrization (OTFP) temperature-accelerated molecular dynamics (TAMD) was also used to calculate two-dimensional free energy surfaces (FESs) related to single/double Na+ translocation through the SF of the homology-based Nav1.2 model and the cryo-EM Nav1.2 structure, with different realizations of the DEKA filter domain. These additional simulations revealed distinct mechanisms for single and double Na+ permeation through the wild-type SF, which has a charged lysine in the DEKA ring. Moreover, the configurations of the ions in the SF corresponding to the metastable states of the FESs are specific for each SF motif. Overall, the description of these mechanisms gives us new insights into ion conduction in human Nav cryo-EM-based and cryo-EM configurations that could advance understanding of these systems and how they differ from potassium and bacterial Nav channels.

Molecular determinants of μ-conotoxin KIIIA interaction with the human voltage-gated sodium channel NaV1.7.

The voltage-gated sodium (NaV) channel subtype NaV1.7 plays a critical role in pain signaling, making it an important drug target. Here we studied the molecular interactions between μ-Conotoxin KIIIA (KIIIA) and the human NaV1.7 channel (hNaV1.7). We developed a structural model of hNaV1.7 using Rosetta computational modeling and performed in silico docking of KIIIA using RosettaDock to predict residues forming specific pairwise contacts between KIIIA and hNaV1.7. We experimentally validated these contacts using mutant cycle analysis. Comparison between our KIIIA-hNaV1.7 model and the cryo-EM structure of KIIIA-hNaV1.2 revealed key similarities and differences between NaV channel subtypes with potential implications for the molecular mechanism of toxin block. The accuracy of our integrative approach, combining structural data with computational modeling, experimental validation, and molecular dynamics simulations, suggests that Rosetta structural predictions will be useful for rational design of novel biologics targeting specific NaV channels.

Beyond CBD: Inhibitory effects of lesser studied phytocannabinoids on human voltage-gated sodium channels.

Introduction: Cannabis contains cannabidiol (CBD), the main non-psychoactive phytocannabinoid, but also many other phytocannabinoids that have therapeutic potential in the treatment of epilepsy. Indeed, the phytocannabinoids cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabichromenic acid (CBCA) and cannabichromene (CBC) have recently been shown to have anti-convulsant effects in a mouse model of Dravet syndrome (DS), an intractable form of epilepsy. Recent studies demonstrate that CBD inhibits voltage-gated sodium channel function, however, whether these other anti-convulsant phytocannabinoids affect these classic epilepsy drug-targets is unknown. Voltage-gated sodium (NaV) channels play a pivotal role in initiation and propagation of the neuronal action potential and NaV1.1, NaV1.2, NaV1.6 and NaV1.7 are associated with the intractable epilepsies and pain conditions. Methods: In this study, using automated-planar patch-clamp technology, we assessed the profile of the phytocannabinoids CBGA, CBDVA, cannabigerol (CBG), CBCA and CBC against these human voltage-gated sodium channels subtypes expressed in mammalian cells and compared the effects to CBD. Results: CBD and CBGA inhibited peak current amplitude in the low micromolar range in a concentration-dependent manner, while CBG, CBCA and CBC revealed only modest inhibition for this subset of sodium channels. CBDVA inhibited NaV1.6 peak currents in the low micromolar range in a concentration-dependent fashion, while only exhibiting modest inhibitory effects on NaV1.1, NaV1.2, and NaV1.7 channels. CBD and CBGA non-selectively inhibited all channel subtypes examined, whereas CBDVA was selective for NaV1.6. In addition, to better understand the mechanism of this inhibition, we examined the biophysical properties of these channels in the presence of each cannabinoid. CBD reduced NaV1.1 and NaV1.7 channel availability by modulating the voltage-dependence of steady-state fast inactivation (SSFI, V0.5 inact), and for NaV1.7 channel conductance was reduced. CBGA also reduced NaV1.1 and NaV1.7 channel availability by shifting the voltage-dependence of activation (V0.5 act) to a more depolarized potential, and for NaV1.7 SSFI was shifted to a more hyperpolarized potential. CBDVA reduced channel availability by modifying conductance, SSFI and recovery from SSFI for all four channels, except for NaV1.2, where V0.5 inact was unaffected. Discussion: Collectively, these data advance our understanding of the molecular actions of lesser studied phytocannabinoids on voltage-gated sodium channel proteins.

Using rosetta and alphafold to assess Jingzhaotoxin-III binding to hNav1.5 and hKv2.1.

Ion channels are pore-forming transmembrane proteins regulating ion flux across cell membranes and regulators of the ventricular action potential. The human voltage-gated sodium channel 1.5 (hNav1.5) is predominantly expressed in cardiac myocytes and is solely responsible for the ventricular action potential's initial upstroke phase. A series of mutations in hNav1.5 are classified as Long QT Syndrome subtype 3 (LQT3) and are linked to hNav1.5 gain-of-function, resulting in an enhanced inward current prolonging repolarization. Alternatively, improper hNav1.5 inactivation results in a prolonged, late sodium current during the action potential plateau, causing a similar LQT3 gain-of function phenotype. Current drug therapies for hNav1.5-associated arrhythmia involve combinations of beta-adrenergic blockers and hNav1.5-targeting small-molecules such as flecainide. Alternatively, hNav1.5-targeting peptide toxins have the potential as novel therapeutics and investigational tools for ion channel disorders. One such peptide, Jingzhaotoxin-III (JzTx-III) is a 36-residue peptide that has been found to inhibit hNav1.5 and human voltage-gated potassium channel isoform 2.1 (hKv2.1) but does not inhibit neuronal and skeletal hNav channels. Mutational studies of JzTx-III identified unique residues that modulate hNav1.5 and hKv2.1 activity. We hypothesize that prior single-point mutational data will demonstrate differing sets of binding free energies and peptide-channel contacts for hNav1.5 and hKv2.1. In this study, we used AlphaFold and Rosetta to generate models of hNav1.5 and hKv2.1 in the deactivated state, docked JzTx-III to each channel, and assessed in silico binding free energies of wild-type and single-point mutations. This structural and energetic information can then be used as a guideline for multi-point mutational design to predict improved hNav1.5 binding specificity for further investigation of LQTS and pro-arrhythmic ventricular action potentials.

Simulations reveal molecular mechanisms of high affinity and selective binding of the tarantula venom protoxin-2 to the human NaV1.7 channel.

Human voltage-gated sodium channels (hNaV) are responsible for initiating and propagating action potentials in excitable cells and have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels, present at the endings of pain-sensing nerves, have garnered a lot of scientific interest as potential targets for pain therapeutics by channel inhibition. Certain natural peptide toxins, such as the tarantula protoxin-2 (ProTx2), have high selectivity for hNaV1.7, and hence they serve as valuable scaffolds for peptide design in pain therapeutics. Here, we present the mechanisms by which ProTx2 can bind and modulate hNaV1.7 channel gating kinetics. Using Rosetta biomolecular modeling software, we constructed atomistic structural models of the hNaV1.7 Voltage-Sensing Domain (VSD) 2 and 4 in the activated and deactivated states. Then, we docked ProTx2 onto the VSDs, embedded the structures into lipid bilayers solvated by aqueous NaCl solution, and performed microsecond-long all-atom molecular dynamics (MD) simulations of those systems using the CHARMM36m force field and Amber PMEMD software. Afterward, we performed binding energetics analysis using molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations and conducted Brownian Dynamics simulations using BrownDye to calculate the second-order association rate constants. Our analysis revealed numerous important inter-residue contacts that contribute to the toxin's high affinity for the channel. By understanding the various state- and subtype-specific mechanisms of toxin binding to the channel, we can develop strategies to improve the selectivity and potency of peptide toxins to create effective and safe pain therapeutics. In addition, the results could be incorporated into functional kinetic models to elucidate how ProTx2 can modulate electrical signal propagation in dorsal root ganglion neurons and thus pain sensing.

Cryo-EM structure of human voltage-gated sodium channel Nav1.6

Voltage-gated sodium channel Nav1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Nav1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Nav1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Nav1.6 in the presence of auxiliary subunits β1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the β1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Nav1.6-specific blockers. Structural mapping of disease-related Nav1.6 mutations provides insights into their pathogenic mechanism.

Exploring the Pivotal Components Influencing the Side Effects Induced by an Analgesic-Antitumor Peptide from Scorpion Venom on Human Voltage-Gated Sodium Channels 1.4 and 1.5 through Computational Simulation.

Voltage-gated sodium channels (VGSCs, or Nav) are important determinants of action potential generation and propagation. Efforts are underway to develop medicines targeting different channel subtypes for the treatment of related channelopathies. However, a high degree of conservation across its nine subtypes could lead to the off-target adverse effects on skeletal and cardiac muscles due to acting on primary skeletal muscle sodium channel Nav1.4 and cardiac muscle sodium channel Nav1.5, respectively. For a long evolutionary process, some peptide toxins from venoms have been found to be highly potent yet selective on ion channel subtypes and, therefore, hold the promising potential to be developed into therapeutic agents. In this research, all-atom molecular dynamic methods were used to elucidate the selective mechanisms of an analgesic-antitumor β-scorpion toxin (AGAP) with human Nav1.4 and Nav1.5 in order to unravel the primary reason for the production of its adverse reactions on the skeletal and cardiac muscles. Our results suggest that the rational distribution of residues with ring structures near position 38 and positive residues in the C-terminal on AGAP are critical factors to ensure its analgesic efficacy. Moreover, the substitution for residues with benzene is beneficial to reduce its side effects.

The Deadly Toxin Arsenal of the Tree-Dwelling Australian Funnel-Web Spiders.

Australian funnel-web spiders are amongst the most dangerous venomous animals. Their venoms induce potentially deadly symptoms, including hyper- and hypotension, tachycardia, bradycardia and pulmonary oedema. Human envenomation is more frequent with the ground-dwelling species, including the infamous Sydney funnel-web spider (Atrax robustus); although, only two tree-dwelling species induce more severe envenomation. To unravel the mechanisms that lead to this stark difference in clinical outcomes, we investigated the venom transcriptome and proteome of arboreal Hadronyche cerberea and H. formidabilis. Overall, Hadronyche venoms comprised 44 toxin superfamilies, with 12 being exclusive to tree-dwellers. Surprisingly, the major venom components were neprilysins and uncharacterized peptides, in addition to the well-known ω- and δ-hexatoxins and double-knot peptides. The insecticidal effects of Hadronyche venom on sheep blowflies were more potent than Atrax venom, and the venom of both tree- and ground-dwelling species potently modulated human voltage-gated sodium channels, particularly NaV1.2. Only the venom of tree-dwellers exhibited potent modulation of voltage-gated calcium channels. H. formidabilis appeared to be under less diversifying selection pressure compared to the newly adapted tree-dweller, H. cerberea. Thus, this study contributes to unravelling the fascinating molecular and pharmacological basis for the severe envenomation caused by the Australian tree-dwelling funnel-web spiders.

µ-Conotoxins Targeting the Human Voltage-Gated Sodium Channel Subtype NaV1.7

µ-Conotoxins are small, potent, peptide voltage-gated sodium (NaV) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype NaV1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human NaV1.7 (hNaV1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different NaV channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNaV channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNaV1.7. Analysis of other µ-conotoxins at hNaV1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNaV1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNaV1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNaV1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNaV1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNaV1.7.

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.

Determination of the toxicity equivalency factors for ciguatoxins using human sodium channels

Ciguatoxins (CTXs) which are produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa and share a ladder-shaped polyether structure, are causative compounds of one of the most frequent foodborne illness disease known as ciguatera fish poisoning (CFP). CFP was initially found in tropical and subtropical areas but nowadays the dinoflagellates producers of ciguatoxins had spread to European coasts. Therefore, this raises the need of establishing toxicity equivalency factors for the different compounds that can contribute to ciguatera fish poisoning, since biological methods have been replaced by analytical techniques. Thus, in this work, the effects of six compounds causative of ciguatera, on their main target, the human voltage-gated sodium channels have been analyzed for the first time. The results presented here led to the conclusion that the order of potency was CTX1B, CTX3B, CTX4A, gambierol, gambierone and MTX3. Furthermore, the data indicate that the activation voltage of sodium channels is more sensitive to detect ciguatoxins than their effect on the peak sodium current amplitude.

Accelerated Production and Maturation of Human Induced Pluripotent Stem Cell Derived Nociceptors

The scarce availability and prohibitive costs of primary human DRG tissues limits the scalability of in vitro models for pain drug discovery and disease modeling. Human induced pluripotent stem cell (hiPSC)-derived sensory neurons provide a near-limitless amount of tissue for these applications. Using an accelerated, step-wise, directed differentiation protocol that proceeds through a novel ‘primal ectoderm’ population, we are able to manufacture nociceptors rapidly and at scale by relying on a simplified chemically-defined basal medium, recombinant growth factors and small molecules. Following production and only one week of maturation, these nociceptors have a consistent molecular phenotype and express key therapeutic pain targets such as human voltage-gated sodium channels Nav1.7, 1.8, and 1.9, which are primarily responsible for eliciting nociceptor action potentials, as well as the human capsaicin receptor TRPV1. Using whole-cell patch clamp electrophysiology, we demonstrate that these neurons fire action potentials and possess burst-firing ability by day 9 in vitro as assessed by performing stepwise depolarizing current injections in current clamp mode. We also show these neurons exhibit inward and outward currents in response to voltage steps in voltage clamp mode. We have also demonstrated that these neurons are responsive to ligands such as capsaicin using calcium imaging region-of-interest analysis. Greater than 30% of neuronal cell bodies respond to 100 nM capsaicin following an additional 2 to 3 weeks of maturation. Importantly, this response was ablated by capsazepine, thus confirming response specificity. These data demonstrate that hiPSC nociceptors have the potential to screen and identify novel analgesic or anti-nociceptive compounds for use in humans.

A novel familial SCN5A exon 20 deletion is associated with a heterogeneous phenotype

The SCN5A gene, located on chromosome 3p21, has 28 exons and is a member of the human voltage-gated sodium channel gene family. Genetic variation in SCN5A is associated with a diverse range of phenotypes. Due to incomplete penetrance, delayed expression, inherent low signal-to-noise ratio, and marked phenotypic heterogeneity, rare novel variants in SCN5A could be misinterpreted. Hence, defining the phenotypic characteristics of these rare SCN5A variants in humans is of importance. We describe the phenotypic heterogeneity noted in 4 familial carriers of a rare, previously unreported, large deletion in exon 20 of SCN5A (c.3667-?_c.3840C +?del) and discuss the mechanisms that underlie this heterogeneity.

Comparative structural analysis of human Nav1.1 and Nav1.5 reveals mutational hotspots for sodium channelopathies

Among the nine subtypes of human voltage-gated sodium (Nav) channels, the brain and cardiac isoforms, Nav1.1 and Nav1.5, each carry more than 400 missense mutations respectively associated with epilepsy and cardiac disorders. High-resolution structures are required for structure-function relationship dissection of the disease variants. We report the cryo-EM structures of the full-length human Nav1.1-β4 complex at 3.3 Å resolution here and the Nav1.5-E1784K variant in the accompanying paper. Up to 341 and 261 disease-related missense mutations in Nav1.1 and Nav1.5, respectively, are resolved. Comparative structural analysis reveals several clusters of disease mutations that are common to both Nav1.1 and Nav1.5. Among these, the majority of mutations on the extracellular loops above the pore domain and the supporting segments for the selectivity filter may impair structural integrity, while those on the pore domain and the voltage-sensing domains mostly interfere with electromechanical coupling and fast inactivation. Our systematic structural delineation of these mutations provides important insight into their pathogenic mechanism, which will facilitate the development of precise therapeutic interventions against various sodium channelopathies.

Engineering of highly potent and selective HNTX-III mutant against hNav1.7 sodium channel for treatment of pain

Human voltage-gated sodium channel Nav1.7 (hNav1.7) is involved in the generation and conduction of neuropathic and nociceptive pain signals. Compelling genetic and preclinical studies have validated that hNav1.7 is a therapeutic target for the treatment of pain; however, there is a dearth of currently available compounds capable of targeting hNav1.7 with high potency and specificity. Hainantoxin-III (HNTX-III) is a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. Here, we report the engineering of improved potency and Nav selectivity of hNav1.7 inhibition peptides derived from the HNTX-III scaffold. Alanine scanning mutagenesis showed key residues for HNTX-III interacting with hNav1.7. Site-directed mutagenesis analysis indicated key residues on hNav1.7 interacting with HNTX-III. Molecular docking was conducted to clarify the binding interface between HNTX-III and Nav1.7 and guide the molecular engineering process. Ultimately, we obtained H4 [K0G1-P18K-A21L-V] based on molecular docking of HNTX-III and hNav1.7 with a 30-fold improved potency (IC50 0.007 ± 0.001 μM) and >1000-fold selectivity against Nav1.4 and Nav1.5. H4 also showed robust analgesia in the acute and chronic inflammatory pain model and neuropathic pain model. Thus, our results provide further insight into peptide toxins that may prove useful in guiding the development of inhibitors with improved potency and selectivity for Nav subtypes with robust analgesia.

Functional modulation of the human voltage-gated sodium channel NaV1.8 by auxiliary β subunits

ABSTRACT The voltage-gated sodium channel Nav1.8 mediates the tetrodotoxin-resistant (TTX-R) Na+ current in nociceptive primary sensory neurons, which has an important role in the transmission of painful stimuli. Here, we describe the functional modulation of the human Nav1.8 α-subunit in Xenopus oocytes by auxiliary β subunits. We found that the β3 subunit down-regulated the maximal Na+ current amplitude and decelerated recovery from inactivation of hNav1.8, whereas the β1 and β2 subunits had no such effects. The specific regulation of Nav1.8 by the β3 subunit constitutes a potential novel regulatory mechanism of the TTX-R Na+ current in primary sensory neurons with potential implications in chronic pain states. In particular, neuropathic pain states are characterized by a down-regulation of Nav1.8 accompanied by increased expression of the β3 subunit. Our results suggest that these two phenomena may be correlated, and that increased levels of the β3 subunit may directly contribute to the down-regulation of Nav1.8. To determine which domain of the β3 subunit is responsible for the specific regulation of hNav1.8, we created chimeras of the β1 and β3 subunits and co-expressed them with the hNav1.8 α-subunit in Xenopus oocytes. The intracellular domain of the β3 subunit was shown to be responsible for the down-regulation of maximal Nav1.8 current amplitudes. In contrast, the extracellular domain mediated the effect of the β3 subunit on hNav1.8 recovery kinetics.