Sodium Channel Research Articles

Molecular determinants of resurgent sodium currents mediated by Navβ4 peptide and A-type FHFs.

Resurgent current (INaR ) generated by voltage-gated sodium channels (VGSCs) plays an essential role in maintaining high-frequency firing of many neurons and contributes to disease pathophysiology such as epilepsy and painful disorders. Targeting INaR may present a highly promising strategy in the treatment of these diseases. Navβ4 and A-type fibroblast growth factor homologous factors (FHFs) have been identified as two classes of important INaR mediators; however, their receptor sites in VGSCs remain unknown, which hinders the development of novel agents to effectively target INaR . Navβ4 and FHF4A can mediate INaR generation through the amino acid segment located in their C-terminus and N-terminus, respectively. We mainly employed site-directed mutagenesis, chimera construction and whole-cell patch-clamp recording to explore the receptor sites of Navβ4 peptide and FHF4A in Nav1.7 and Nav1.8. We show that the receptor of Navβ4-peptide involves four residues, N395, N945, F1737 and Y1744, in Nav1.7 DI-S6, DII-S6, and DIV-S6. We show that A-type FHFs generating INaR depends on the segment located at the very beginning, not at the distal end, of the FHF4 N-terminus domain. We show that the receptor site of A-type FHFs also resides in VGSC inner pore region. We further show that an asparagine at DIIS6, N891 in Nav1.8, is a major determinant of INaR generated by A-type FHFs in VGSCs. Cryo-EM structures reveal that the side chains of the critical residues project into the VGSC channel pore. Our findings provide additional evidence that Navβ4 peptide and A-type FHFs function as open-channel pore blockers and highlight channel inner pore region as a hotspot for development of novel agents targeting INaR .

Unraveling the dynamics of firing patterns for neurons with impairment of sodium channels.

Various factors such as mechanical trauma, chemical trauma, local ischemia, and inflammation can impair voltage-gated sodium channels (Nav) in neurons. These impairments lead to a distinctive leftward shift in the activation and inactivation curves of voltage-gated sodium channels. The resulting sodium channel impairments in neurons are known to affect firing patterns, which play a significant role in neuronal activities within the nervous system. However, the underlying dynamic mechanism for the emergence of these firing patterns remains unclear. In this study, we systematically investigated the effects of sodium channel dysfunction on individual neuronal dynamics and firing patterns. By employing codimension-1 bifurcation analysis, we revealed the underlying dynamical mechanism responsible for the generation of different firing patterns. Additionally, through codimension-2 bifurcation analysis, we theoretically determined the distribution of firing patterns on different parameter planes. Our results indicate that the firing patterns of impaired neurons are regulated by multiple parameters, with firing pattern transitions caused by the degree of sodium channel impairment being more diverse than those caused by the ratio of impaired sodium channel and current. Furthermore, we observed that the firing pattern of tonic firing is more likely to be the norm in impaired sodium channel neurons, providing valuable insights into the signaling of impaired neurons. Overall, our findings highlight the intricate relationships among sodium channel impairments, neuronal dynamics, and firing patterns, shedding light on the impact of disruptions in ion concentration gradients on neuronal function.

Comparison of Nocifensive Behavior in NaV1.7-, NaV1.8-, and NaV1.9-Channelrhodopsin-2 Mice by Selective Optogenetic Activation of Targeted Sodium Channel Subtype-Expressing Afferents.

Voltage-gated sodium channels, including NaV1.7, NaV1.8, and NaV1.9, play important roles in pain transmission and chronic pain development. However, the specific mechanisms of their action remain unclear, highlighting the need for invivo stimulation studies of these channels. Optogenetics, a novel technique for targeting the activation or inhibition of specific neural circuits using light, offers a promising solution. In our previous study, we used optogenetics to selectively excite NaV1.7-expressing neurons in the dorsal root ganglion of mice to induce nocifensive behavior. Here, we further characterize the impact of nocifensive behavior by activation of NaV1.7, NaV1.8, or NaV1.9-expressing neurons. Using CRISPR/Cas9-mediated homologous recombination, NaV1.7-iCre, NaV1.8-iCre, or NaV1.9-iCre mice expressing iCre recombinase under the control of the endogenous NaV1.7, NaV1.8, or NaV1.9 gene promoter were produced. These mice were then bred with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7-ChR2, NaV1.8-ChR2, or NaV1.9-ChR2 mice. Blue light exposure triggered paw withdrawal in all mice, with the strongest response in NaV1.8-ChR2 mice. These light sensitivity differences observed across NaV1.x-ChR2 mice may be dependent on ChR2 expression or reflect the inherent disparities in their pain transmission roles. In conclusion, we have generated noninvasive pain models, with optically activated peripheral nociceptors. We believe that studies using optogenetics will further elucidate the role of sodium channel subtypes in pain transmission.

Methylcobalamin as a candidate for chronic peripheral neuropathic pain therapy: review of molecular pharmacology actiona.

Chronic peripheral neuropathic pain therapy currently focuses on modulating neuroinflammatory conditions. Methylcobalamin (MeCbl), a neuroregenerative agent, modulates neuroinflammation. This review aimed to explore the molecular pharmacology action of MeCbl as a chronic peripheral neuropathic pain therapeutic agent. MeCbl plays a role in various cellular processes and may have therapeutic potential in neurodegenerative diseases. Intracellular MeCbl modulates inflammation by regulating the activity of T lymphocytes and natural killer cells as well as secretion of inflammatory cytokines, namely, tumor necrosis factor-α, interleukin-6, interleukin-1β, epidermal growth factor, and neuronal growth factor. MeCbl can reduce pain symptoms in chronic neuropathic pain conditions by decreasing excitation and hyperpolarization-induced ion channel activity in medium-sized dorsal root ganglion (DRG) neurons and the expression of transient receptor potential ankyrin 1, transient receptor potential cation channel subfamily M member 8, phosphorylated p38MAPK, transient receptor potential cation channel subfamily V members 1 and 4 in the DRG, and the voltage-gated sodium channel in axons.

Early developmental alterations of CA1 pyramidal cells in Dravet syndrome

Dravet Syndrome (DS) is most often caused by heterozygous loss-of-function mutations in the voltage-gated sodium channel gene SCN1A (Nav1.1), resulting in severe epilepsy and neurodevelopmental impairment thought to be cause by reduced interneuron excitability. However, recent studies in mouse models suggest that interneuron dysfunction alone does not completely explain all the cellular and network impairments seen in DS. Here, we investigated the development of the intrinsic, synaptic, and network properties of CA1 pyramidal cells in a DS model prior to the appearance of overt seizures. We report that CA1 pyramidal cell development is altered by heterozygous reduction of Scn1a, and propose that this is explained by a period of reduced intrinsic excitability in early postnatal life, during which Scn1a is normally expressed in hippocampal pyramidal cells. We also use a novel ex vivo model of homeostatic plasticity to show an instability in homeostatic response during DS epileptogenesis. This study provides evidence for the early effects of Scn1a haploinsufficiency in pyramidal cells in contributing to the pathophysiology of DS.

Central Neuropathic Pain

ABSTRACT OBJECTIVE This article provides an approach to the assessment, diagnosis, and treatment of central neuropathic pain. LATEST DEVELOPMENTS Recent studies of the pathophysiology of central neuropathic pain, including evidence of changes in the expression of voltage-gated sodium channels and N-methyl-d-aspartate (NMDA) receptors, may provide the basis for new therapies. Other areas of current research include the role of cannabinoid-receptor activity and microglial cell activation in various animal models of central neuropathic pain. New observations regarding changes in primary afferent neuronal activity in central neuropathic pain and the preliminary observation that peripheral nerve blocks may relieve pain due to central neuropathic etiologies provide new insights into both the mechanism and treatment of central neuropathic pain. ESSENTIAL POINTS In the patient populations treated by neurologists, central neuropathic pain develops most frequently following spinal cord injury, multiple sclerosis, or stroke. A multimodal, individualized approach to the management of central neuropathic pain is necessary to optimize pain relief and may require multiple treatment trials to achieve the best outcome.

NaV1.1 contributes to the cell cycle of human mesenchymal stem cells by regulating AKT and CDK2.

Non-excitable cells express sodium voltage-gated channel alpha subunit 1 gene and protein (known as SCN1A and NaV1.1, respectively); however, the functions of NaV1.1 are unclear. In this study, we investigated the role of SCN1A and NaV1.1 in human mesenchymal stem cells (MSCs). We found that SCN1A was expressed in MSCs, and abundant expression of NaV1.1 was observed in the endoplasmic reticulum; however, this expression was not found to be related to Na+ currents. SCN1A-silencing reduced MSC proliferation and delayed the cell cycle in the S phase. SCN1A silencing also suppressed the protein levels of CDK2 and AKT (herein referring to total AKT), despite similar mRNA expression, and inhibited AKT phosphorylation in MSCs. A cycloheximide-chase assay showed that SCN1A-silencing induced CDK2 but not AKT protein degradation in MSCs. A proteolysis inhibition assay using epoxomicin, bafilomycin A1 and NH4Cl revealed that both the ubiquitin-proteasome system and the autophagy and endo-lysosome system were irrelevant to CDK2 and AKT protein reduction in SCN1A-silenced MSCs. The AKT inhibitor LY294002 did not affect the degradation and nuclear localization of CDK2 in MSCs. Likewise, the AKT activator SC79 did not attenuate the SCN1A-silencing effects on CDK2 in MSCs. These results suggest that NaV1.1 contributes to the cell cycle of MSCs by regulating the post-translational control of AKT and CDK2.

Renal Kallikrein-1 Contributes to Cleavage of Gamma-Epithelial Sodium Channel (ENaC) in Response to a Short-Term, Low-Sodium, High-Potassium Diet

Renal Kallikrein-1 Contributes to Cleavage of Gamma-Epithelial Sodium Channel (ENaC) in Response to a Short-Term, Low-Sodium, High-Potassium Diet

Regulating distal nephron functions and salt sensitivity.

This review highlights the molecular basis of salt sensitivity in hypertension, with a focus on the regulation of sodium transport in the distal nephron. Sodium reabsorption in this region is often linked to the actions of aldosterone, although in recent years numerous findings have been reported on the aldosterone-independent pathway of acquiring salt sensitivity by potassium deficiency or potassium loading. The key to this discussion is the interplay, through extracellular potassium concentration, between the first part of the tubules expressing the Na+-Cl- cotransporter (NCC) and the second part expressing the epithelial Na+ channel (ENaC). The molecular pathways such as with-no-lysine 1 (WNK)-STE20/SPS1-related proline-alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) signaling, Kelch-like family member 3 (KLHL3)-cullin 3 (CUL3) complex, protein phosphatases, and mechanistic target of rapamycin complex 2 (mTORC2)-Nedd4L pathway are described as the mechanism by which salt sensitivity on blood pressure is acquired in response to changes in physiological conditions including potassium depletion or loading. This review highlights the potential for targeting these molecular pathways to develop novel therapeutic strategies for the treatment of salt-sensitive hypertension, the mechanism of which remains to be elucidated.

Discovery of E0199: A novel compound targeting both peripheral NaV and KV7 channels to alleviate neuropathic pain

This research study focuses on addressing the limitations of current neuropathic pain (NP) treatments by developing a novel dual-target modulator, E0199, targeting both NaV1.7, NaV1.8, and NaV1.9 and KV7 channels, a crucial regulator in controlling NP symptoms. The objective of the study was to synthesize a compound capable of modulating these channels to alleviate NP. Through an experimental design involving both in vitro and in vivo methods, E0199 was tested for its efficacy on ion channels and its therapeutic potential in a chronic constriction injury (CCI) mouse model. The results demonstrated that E0199 significantly inhibited NaV1.7, NaV1.8, and NaV1.9 channels with a particularly low half maximal inhibitory concentration (IC50) for NaV1.9 by promoting sodium channel inactivation, and also effectively increased KV7.2/7.3, KV7.2, and KV7.5 channels, excluding KV7.1 by promoting potassium channel activation. This dual action significantly reduced the excitability of dorsal root ganglion neurons and alleviated pain hypersensitivity in mice at low doses, indicating a potent analgesic effect without affecting heart and skeletal muscle ion channels critically. The safety of E0199 was supported by neurobehavioral evaluations. Conclusively, E0199 represents a ground-breaking approach in NP treatment, showcasing the potential of dual-target small-molecule compounds in providing a more effective and safe therapeutic option for NP. This study introduces a promising direction for the future development of NP therapeutics.

Prostaglandin E2 Receptor 4 Contributes to Regulating Epithelial Sodium Channel (ENaC) Activity in the Kidney Collecting Duct

Prostaglandin E2 Receptor 4 Contributes to Regulating Epithelial Sodium Channel (ENaC) Activity in the Kidney Collecting Duct

Different taste map for amiloride sensitivity, response frequency, and threshold to NaCl in the rostral nucleus of the solitary tract in rats.

Studies on taste bud cells and brain stem relay nuclei suggest that alternative pathways convey information regarding different taste qualities. Building on the hypothesis that amiloride (epithelial Na channel antagonist)-sensitive neurons respond to palatable salt (low-concentration) and amiloride-insensitive neurons respond to aversive salt (high-concentration), we investigated the histological distribution of taste-sensitive neurons in the rostral nucleus of the solitary tract in rats and their NaCl and amiloride sensitivities. We recorded neuronal activity in extracellular single units using multi-barrel glass micropipettes and reconstructed their locations on the rostrocaudal and mediolateral axes. Seventy-three taste-sensitive neurons were categorized into the best-taste category. The amiloride sensitivities of the 31 neurons were examined for 0.1, 0.2, 0.4, and 0.8 M NaCl. The neuronal distribution of amiloride-sensitive neurons was located in the lateral region, while amiloride-insensitive neurons were located in the medial region. The amiloride-sensitive neurons responded to low salt concentrations, signaling the NaCl levels required by body fluids. Amiloride-insensitive neurons were silent at low salt concentrations but may function as warning signals for high salt concentrations. Low-threshold and/or high-response neurons were located in the rostrolateral region. In contrast, high-threshold and/or low-response neurons were located in the caudal-medial region.

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, designated Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch–clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

TRPV4 Channel Modulators as Potential Drug Candidates for Cystic Fibrosis.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in defective chloride ion channels. This leads to thick, dehydrated mucus that severely disrupts mucociliary clearance in the respiratory system and triggers infection that eventually is the cause of death of CF patients. Current therapeutic strategies primarily focus on restoring CFTR function, blocking epithelial sodium channels to prevent mucus dehydration, or directly targeting mucus to reduce its viscosity. Among the ion channels expressed in ciliated bronchial epithelial cells, the transient receptor potential vanilloid 4 (TRPV4) channel emerges as a significant channel in CF pathogenesis. Activation of TRPV4 channels affects the regulation of airway surface liquid by modulating sodium absorption and intracellular calcium levels, which indirectly influences CFTR activity. TRPV4 is also involved in the regulatory volume decrease (RVD) process and enhances inflammatory responses in CF patients. Here, we combine current findings on TRPV4 channel modulation as a promising therapeutic approach for CF. Although limited studies have directly explored TRPV4 in CF, emerging evidence indicates that TRPV4 activation can significantly impact key pathological processes in the disease. Further investigation into TRPV4 modulators could lead to innovative treatments that alleviate severe respiratory complications and improve outcomes for CF patients.

Structural basis for the rescue of hyperexcitable cells by the amyotrophic lateral sclerosis drug Riluzole

Neuronal hyperexcitability is a key element of many neurodegenerative disorders including the motor neuron disease Amyotrophic Lateral Sclerosis (ALS), where it occurs associated with elevated late sodium current (INaL). INaL results from incomplete inactivation of voltage-gated sodium channels (VGSCs) after their opening and shapes physiological membrane excitability. However, dysfunctional increases can cause hyperexcitability-associated diseases. Here we reveal the atypical binding mechanism which explains how the neuroprotective ALS-treatment drug riluzole stabilises VGSCs in their inactivated state to cause the suppression of INaL that leads to reversed cellular overexcitability. Riluzole accumulates in the membrane and enters VGSCs through openings to their membrane-accessible fenestrations. Riluzole binds within these fenestrations to stabilise the inactivated channel state, allowing for the selective allosteric inhibition of INaL without the physical block of Na+ conduction associated with traditional channel pore binding VGSC drugs. We further demonstrate that riluzole can reproduce these effects on a disease variant of the non-neuronal VGSC isoform Nav1.4, where pathologically increased INaL is caused directly by mutation. Overall, we identify a model for VGSC inhibition that produces effects consistent with the inhibitory action of riluzole observed in models of ALS. Our findings will aid future drug design and supports research directed towards riluzole repurposing.

Role of protein domains in trafficking and localization of the voltage-gated sodium channel β2 subunit

The voltage-gated sodium (NaV) channel is critical for cardiomyocyte function since it is responsible for action potential initiation and its propagation throughout the cell. It consists of a protein complex made of a pore forming α subunit and associated β subunits, which regulate α subunit function and subcellular localization. We previously showed the implication of N-linked glycosylation and S-acylation of β2 in its polarized trafficking. Here, we present evidence of β2 dimerization. Moreover, we demonstrate the implication of the cytoplasmic tail, extracellular loop, and transmembrane domain on proper β2 folding and export to the cell surface of polarized Madin-Darby canine kidney cells. Substantial alteration, or lack of any of these domains, leads to accumulation of β2 in the endoplasmic reticulum, along with impaired complex N-glycosylation, which is needed for its efficient surface delivery. We also show that these alterations to β2 affected to certain extent NaV1.5 surface localization. Conversely, however, NaV1.5 had little or no influence on β2 trafficking, its localization to the surface, or homodimer formation. Altogether, our data link the architecture of the β2 domains to the establishment of its proper subcellular localization. These findings could provide valuable insights to gain a deeper comprehension of the elusive biology of β subunits in excitable cells, such as neurons and cardiomyocytes.

Study on the cross-resistance of Aedes albopictus (Skuse) (Diptera: Culicidae) to deltamethrin and pyriproxyfen

BackgroundInsecticide resistance poses a significant challenge in the implementation of vector-borne disease control strategies. We have assessed the resistance levels of Aedes albopictus to deltamethrin and pyriproxyfen (PPF) in Fujian Province (China) and investigated the correlation between these resistance levels and mutations in the voltage-gated sodium channel (VGSC).MethodsThe WHO bioassay protocol was used to evaluate the resistance coefficient of Ae. albopictus to deltamethrin and PPF, comparing a susceptible population from the Foshan (FS) area with wild populations from the Sanming (SM), Quanzhou (QZ), Zhangzhou (ZZ), Putian (PT) and Fuzhou (FZ) areas in Fujian Province. Genomic DNA was analyzed by PCR and sequencing to detect knockdown resistance (kdr) in the VGSC, specifically at the pyrethroid resistance alleles V1016V, I1532I and F1534F. Molecular docking was also performed to analyze the binding interactions of PPF and its metabolite 4'-OH-PPF to cytochrome P450 (CYP) 2C19, 2C9 and 3A4 and Ae. albopictus methoprene-tolerant receptors (AeMet), respectively.ResultsThe analysis of resistance to deltamethrin and PPF among Ae. albopictus populations from the various regions revealed that except for the sensitive population in FS and the SM population, the remaining four regional populations demonstrated resistance levels ranging from 4.31- to 18.87-fold for deltamethrin and from 2.85– to 3.62-fold for PPF. Specifically, the FZ and PT populations exhibited high resistance to deltamethrin, whereas the ZZ and QZ populations approached moderate resistance levels. Also, the resistance of the FZ, PT and ZZ populations to PPF increased slowly but consistently with the increasing trend of deltamethrin resistance. Genomic analysis identified multiple non-synonymous mutations within the VGSC gene; the F1534S and F1534L mutations showed significant resistance to deltamethrin in Ae. albopictus. Molecular docking results revealed that PPF and its metabolite 4'-OH-PPF bind to the Ae. albopictus AeMet receptor and CYP2C19.ConclusionsThe wild Ae. albopictus populations of Fujian Province showed varying degrees of resistance to deltamethrin and PPF and a trend of cross-resistance to deltamethrin and PPF. Increased vigilance is needed for potential higher levels of cross-resistance, especially in the PT and FZ regions.Graphical

Infrared neuroglial modulation of spinal locomotor networks

Infrared neural stimulation (INS) emerges as a promising tool for stimulating the nervous system by its high spatial precision and absence of the use of exogenous agents into the tissue, which led to the first successful proof of concept in human brain. While neural networks have been the focal point of INS research, this technique is also non cell type specific as it triggers activity in non electrically excitable cells. Despite increasing interest, there remains to demonstrate well defined simultaneous astrocytic and neuronal signals in response to INS. Using calcium imaging, we show that INS has the capacity to initiate calcium signaling in both astrocytes and neurons simultaneously from the rostral lumbar spinal cord, each exhibiting distinct temporal and amplitude characteristics. Importantly, the mechanism underlying infrared-induced neuronal and astrocytic calcium signaling differ, with neuronal activity relying on sodium channels, whereas induced astrocytic signaling is predominantly influenced by extracellular calcium and TRPV4 channels. Furthermore, our findings demonstrate the frequency shift of neuronal calcium oscillations through infrared stimulation. By deepening our understanding in INS fundamentals, this technique holds great promise for advancing neuroscience, deepening our understanding of pathologies, and potentially paving the way for future clinical applications.

Sudden Cardiac Death and Channelopathies: What Lies behind the Clinical Significance of Rare Splice-Site Alterations in the Genes Involved?

Background and objectives: Sudden cardiac death (SCD) is a natural and unexpected death of cardiac origin that occurs within 1 h from the onset of acute symptoms. The major leading causes of SCD are cardiomyopathies and channelopathies. In this review, we focus on channelopathies, inherited diseases caused by mutations affecting genes encoding membrane ion channels (sodium, potassium or calcium channels) or cellular structures that affect Ca2+ availability. The diagnosis of diseases such as long QT, Brugada syndrome, short QT and catecholaminergic polymorphic ventricular tachycardia (CPVT) is still challenging. Currently, genetic testing and next-generation sequencing allow us to identify many rare alterations. However, some non-coding variants, e.g., splice-site variants, are usually difficult to interpret and to classify. Methods: In our review, we searched for splice-site variants of genes involved in channelopathies, focusing on variants of unknown significance (VUSs) registered on ClinVar up to now. Results: The research led to a high number of splice-site VUSs of genes involved in channelopathies, suggesting the performance of deeper studies. Conclusions: In order to interpret the correlation between variants and pathologies, we discuss experimental studies, such as RNA sequencing and functional analysis of proteins. Unfortunately, as these in vitro analyses cannot always be performed, we draw attention to in silico studies as future perspectives in genetics. This review has the aim of discussing the potential methods of detection and interpretation of VUSs, bringing out the need for a future reclassification of variants with currently unknown significance.

BOS-318 treatment enhances elexacaftor-tezacaftor-ivacaftor (ETI)-mediated improvements in airways hydration and mucociliary transport

BackgroundCFTR triple modulator therapy, elexacaftor-tezacaftor-ivacaftor (ETI) has transformed care for people with cystic fibrosis (pwCF) who have eligible mutations. It is, however, not curative. Response to treatment also varies and lung disease although slowed, remains progressive. We have previously demonstrated that inhibition of the epithelial sodium channel (ENaC) by selective furin inhibition offers an alternative, mutation-agnostic approach to enhance airways hydration and restore mucociliary transport (MCT) in CF. Inhibition of furin therefore, offers a potential therapeutic strategy for those ineligible, intolerant or non-responsive to ETI and may provide a further opportunity for clinical benefit for those currently treated with ETI. The aim of this study was to determine the impact of furin inhibition on ETI responses to assess its utility as an adjunct therapy.MethodsDifferentiated primary human CF bronchial epithelial cells (CF HBECs) were treated with the highly selective furin inhibitor, BOS-318 and ETI. Ion channel function was measured using a 24-channel Transepithelial Current Clamp (TECC-24) system and airways surface hydration was investigated by measuring airway surface liquid (ASL) height and MCT rate.ResultsThe presence of BOS-318 had no effect on the ability of ETI to stimulate CFTR-mediated Cl−secretion but contributed a reduced Na+transportviarobust inhibition of ENaC. This altered ion transport profile effected an improved ASL height and MCT rate which were significantly greater than improvements observed with ETI alone, demonstrating the benefits of the dual approach.ConclusionsSelective furin inhibition has potential to further improve clinical outcomes for all pwCF and offers opportunity as an adjunct to improve responses to currently available CFTR modulator therapies.