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Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.

Abstract Objective To evaluate the analgesic potential, safety, tolerability, and pharmacokinetics of VX-150, a pro-drug of a highly selective NaV1.8 inhibitor, in healthy subjects. Design This was a randomized, double-blind, placebo-controlled, crossover study in healthy subjects. Subjects Twenty healthy male subjects with an age of 18–55 years, inclusive, were enrolled. Eligibility was based on general fitness, absence of current or previous medical conditions that could compromise subject safety, and a training assessment of pain tolerance across pain tests to exclude highly tolerant individuals whose tolerance could compromise the ability to detect analgesic responses. All dosed subjects completed the study. Methods Subjects were randomized 1:1 to one of two sequences receiving a single VX-150 dose and subsequently placebo, or vice versa, with at least 7 days between dosing. A battery of pain tests (pressure, electrical stair, [capsaicin-induced] heat, and cold pressor) was administered before dosing and repetitively up to 10 h after dosing, with blood sampling up to 24 h after dosing. Safety was monitored throughout the study. Data were analyzed with a repeated-measures mixed-effects model. Results VX-150 induced analgesia in a variety of evoked pain tests, without affecting subject safety. Significant effects were reported for the cold pressor and heat pain thresholds. Maximum median concentration for the active moiety was 4.30 µg/mL at 4 h after dosing. Conclusion Results of this proof-of-mechanism study are supportive of the potential of VX-150, a highly selective NaV1.8 channel inhibitor, to treat various pain indications.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10−12 to 10−5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

The voltage-gated sodium channel α-subunit genes comprise a highly conserved gene family. Mutations of three of these genes, SCN1A, SCN2A and SCN8A, are responsible for a significant burden of neurological disease. Recent progress in identification and functional characterization of patient variants is generating new insights and novel approaches to therapy for these devastating disorders. Here we review the basic elements of sodium channel function that are used to characterize patient variants. We summarize a large body of work using global and conditional mouse mutants to characterize the in vivo roles of these channels. We provide an overview of the neurological disorders associated with mutations of the human genes and examples of the effects of patient mutations on channel function. Finally, we highlight therapeutic interventions that are emerging from new insights into mechanisms of sodium channelopathies.A substantial burden of neurological disease is related to mutations in three sodium channel genes: SCN1A, SCN2A and SCN8A. In this Review, Meisler and colleagues discuss the neurological disorders associated with these mutations and also the therapeutic opportunities that are being investigated as a result of these recent mechanistic insights.

Voltage-gated sodium （Nav） channels are essential for the rapid upstroke of action potentials and the propa- gation of electrical signals in nerves and muscles. Defects of Nav channels are associated with a variety of channelopathies. More than 1000 disease-related muta- tions have been identified in Nay channels, with Nay1.1 and Nay1.5 each harboring more than 400 mutations. Nay channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Nav chan- nels are required to understand their function and dis- ease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium （Car） channel Carl.1 provides a template for homology-based structural modeling of the evolutionarily related Nay channels. In this Resource article, we summarized all the reported disease-related mutations in human Nav channels, generated a homologous model of human Nay1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Nay channels, the analysis presented here serves as the base framework for mechanistic investi- gation of Nav channelopathies and for potential struc- ture-based drug discovery.

Voltage-gated sodium (Na V ) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na + channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4–S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ∼4.6 Å wide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na + selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level. Mechanism of Na+ channel action The X-ray crystal structure of a voltage-gated sodium channel from Arcobacter butzleri has been determined, with the channel in the closed-pore conformation. Channels of this type initiate electrical signalling in excitable cells and are the molecular targets for many drugs, but the structural basis for their voltage-dependent activation and ion selectivity is not known. The selectivity filter in this sodium channel is found to be quite short, compared with those in open-pore potassium channels, and the voltage-sensor domains and linkers between segments S4 and S5 seem to dilate the central pore by pivoting together.

AbstractBackgroundThe NaV1.8 voltage-gated sodium channel, expressed in peripheral nociceptive neurons, plays a role in transmitting nociceptive signals. The effect of VX-548, an oral, highly selective inhibitor of NaV1.8, on control of acute pain is being studied.MethodsAfter establishing the selectivity of VX-548 for NaV1.8 inhibition in vitro, we conducted two phase 2 trials involving participants with acute pain after abdominoplasty or bunionectomy. In the abdominoplasty trial, participants were randomly assigned in a 1:1:1:1 ratio to receive one of the following over a 48-hour period: a 100-mg oral loading dose of VX-548, followed by a 50-mg maintenance dose every 12 hours (the high-dose group); a 60-mg loading dose of VX-548, followed by a 30-mg maintenance dose every 12 hours (the middle-dose group); hydrocodone bitartrate–acetaminophen (5 mg of hydrocodone bitartrate and 325 mg of acetaminophen every 6 hours); or oral placebo every 6 hours. In the bunionectomy trial, participants were randomly assigned in a 2:2:1:2:2 ratio to receive one of the following over a 48-hour treatment period: oral high-dose VX-548; middle-dose VX-548; low-dose VX-548 (a 20-mg loading dose, followed by a 10-mg maintenance dose every 12 hours); oral hydrocodone bitartrate–acetaminophen (5 mg of hydrocodone bitartrate and 325 mg of acetaminophen every 6 hours); or oral placebo every 6 hours. The primary end point was the time-weighted sum of the pain-intensity difference (SPID) over the 48-hour period (SPID48), a measure derived from the score on the Numeric Pain Rating Scale (range, 0 to 10; higher scores indicate greater pain) at 19 time points after the first dose of VX-548 or placebo. The main analysis compared each dose of VX-548 with placebo.ResultsA total of 303 participants were enrolled in the abdominoplasty trial and 274 in the bunionectomy trial. The least-squares mean difference between the high-dose VX-548 and placebo groups in the time-weighted SPID48 was 37.8 (95% confidence interval [CI], 9.2 to 66.4) after abdominoplasty and 36.8 (95% CI, 4.6 to 69.0) after bunionectomy. In both trials, participants who received lower doses of VX-548 had results similar to those with placebo. Headache and constipation were common adverse events with VX-548.ConclusionsAs compared with placebo, VX-548 at the highest dose, but not at lower doses, reduced acute pain over a period of 48 hours after abdominoplasty or bunionectomy. VX-548 was associated with adverse events that were mild to moderate in severity. (Funded by Vertex Pharmaceuticals; VX21-548-101 and VX21-548-102 ClinicalTrials.gov numbers, NCT04977336 and NCT05034952.)

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Na v 1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.

The human voltage-gated sodium channel, hNaV1.5, is responsible for the rapid upstroke of the cardiac action potential and is target for antiarrhythmic therapy. Despite the clinical relevance of hNaV1.5-targeting drugs, structure-based molecular mechanisms of promising or problematic drugs have not been investigated at atomic scale to inform drug design. Here, we used Rosetta structural modeling and docking as well as molecular dynamics simulations to study the interactions of antiarrhythmic and local anesthetic drugs with hNaV1.5. These calculations revealed several key drug binding sites formed within the pore lumen that can simultaneously accommodate up to two drug molecules. Molecular dynamics simulations identified a hydrophilic access pathway through the intracellular gate and a hydrophobic access pathway through a fenestration between DIII and DIV. Our results advance the understanding of molecular mechanisms of antiarrhythmic and local anesthetic drug interactions with hNaV1.5 and will be useful for rational design of novel therapeutics.

The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.