Resolving artefacts in voltage-clamp experiments with computational modelling: an application to fast sodium current recordings.

Cellular electrophysiology is the foundation of many fields, from basic science in neurology, cardiology, oncology to safety critical applications for drug safety testing, clinical phenotyping, etc. Patch-clamp voltage clamp is the gold standard technique for studying cellular electrophysiology. Yet, the quality of these experiments is not always transparent, which may lead to erroneous conclusions for studies and applications. Here, we have developed a new computational approach that allows us to explain and predict the experimental artefacts in voltage-clamp experiments. The computational model captures the experimental procedure and its inadequacies, including: voltage offset, series resistance, membrane capacitance and (imperfect) amplifier compensations, such as series resistance compensation and supercharging. The computational model was validated through a series of electrical model cell experiments. Using this computational approach, the artefacts in voltage-clamp experiments of cardiac fast sodium current, one of the most challenging currents to voltage clamp, were able to be resolved and explained through coupling the observed current and the simulated membrane voltage, including some typically observed shifts and delays in the recorded currents. We further demonstrated that the typical way of averaging data for current-voltage relationships would lead to biases in the peak current and shifts in the peak voltage, and such biases can be in the same order of magnitude as those differences reported for disease-causing mutations. Therefore, the presented new computational pipeline will provide a new standard of assessing the voltage-clamp experiments and interpreting the experimental data, which may be able to rectify and provide a better understanding of ion channel mutations and other related applications.

An automated planar patch-clamp approach to measure the membrane potential and resting membrane currents in a human cerebrovascular endothelial cell line.

BACKGROUND: The conventional "whole-cell patch-clamp" recording technique is widely used to measure the resting membrane potential (VM) and to dissect the underlying membrane ionic conductances in isolated vascular endothelial cells. NEW METHOD: Herein, we assessed whether the automated patch-clamp (APC) technology, which replaces the traditional patch-pipette with a planar substrate to permit researchers lacking formal training in electrophysiology to generate large amounts of data in a relatively short time, can be used to characterize the bioelectrical activity of vascular endothelial cells. We assessed whether the Port-a-Patch planar patch-clamp system, which is regarded as the smallest electrophysiological rig available on the market, can be used to measure the VM and resting membrane currents in the human cerebrovascular endothelial cell line, hCMEC/D3. COMPARISON WITH EXISTING METHODS: We demonstrated that the Port-a-Patch planar patch-clamp system provides the same values of the resting VM as those provided by the conventional patch-clamp technique. Furthermore, the APC technology provides preliminary data demonstrating that the resting VM of hCMEC/D3 cells is primarily contributed by Cl- and Na+, as demonstrated with the patch-clamp technique for many other endothelial cell types. CONCLUSIONS: The Port-a-Patch planar patch-clamp system can be successfully used to measure the resting VM and the underlying membrane ionic conductances in hCMEC/D3 cells. We envisage that this easy-to-use APC system could also be extremely useful for the investigation of the membrane currents that can be activated by chemical, thermal, and mechanical stimuli in this cell line as well as in other types of isolated vascular endothelial cells.

Analysis of ion transport properties of Glycine max HKT transporters and identifying a regulation of GmHKT1;1 by the non-functional GmHKT1;4.

The HKT transporter plays an important role for plants in response to salt stress, but the transport property of the soybean HKT transporters at the molecular level is still unclear. Here, using Xenopus oocyte as a heterologous expression system and two-electrode voltage-clamp technique, we identified four HKT transporters, GmHKT1;1, GmHKT1;2, GmHKT1;3, and GmHKT1;4, which all belong to type I subfamily, but having distinct ion transport properties. While GmHKT1;1, GmHKT1;2 and GmHKT1;3 function as Na+ transporters, GmHKT1;1 is less selective against K+ than the two other transporters. Astonishingly, GmHKT1;4, which lacks transmembrane segments and has no ion permeability, is significantly expressed, and its gene expression pattern is different from the other three GmHKTs under salt stress. Interestingly, GmHKT1;4 reduced the Na+/K+ currents mediated by GmHKT1;1. Further study showed that the transport ability of GmHKT1;1 regulated by GmHKT1;4 was related to the structural differences in the first intracellular domain and the fourth repeat domain. Overall, we have identified one unique GmHKT member, GmHKT1;4, which modulates the Na+ and K+ transport ability of GmHKT1;1 via direct interaction. Thus, we have revealed a new type of HKTs interaction model for altering their ion transport properties.

Disease-associated missense mutations in the pore loop of polycystin-2 alter its ion channel function in a heterologous expression system.

Polycystin-2 (PC2) is mutated in ∼15% of patients with autosomal dominant polycystic kidney disease (ADPKD). PC2 belongs to the family of transient receptor potential (TRP) channels and can function as a homotetramer. We investigated whether three disease-associated mutations (F629S, C632R, or R638C) localized in the channel's pore loop alter ion channel properties of human PC2 expressed in Xenopus laevis oocytes. Expression of wild-type (WT) PC2 typically resulted in small but measurable Na+ inward currents in the absence of extracellular divalent cations. These currents were no longer observed when individual pore mutations were introduced in WT PC2. Similarly, Na+ inward currents mediated by the F604P gain-of-function (GOF) PC2 construct (PC2 F604P) were abolished by each of the three pore mutations. In contrast, when the mutations were introduced in another GOF construct, PC2 L677A N681A, only C632R had a complete loss-of-function effect, whereas significant residual Na+ inward currents were observed with F629S (∼15%) and R638C (∼30%). Importantly, the R638C mutation also abolished the Ca2+ permeability of PC2 L677A N681A and altered its monovalent cation selectivity. To elucidate the molecular mechanisms by which the R638C mutation affects channel function, molecular dynamics (MD) simulations were used in combination with functional experiments and site-directed mutagenesis. Our findings suggest that R638C stabilizes ionic interactions between Na+ ions and the selectivity filter residue D643. This probably explains the reduced monovalent cation conductance of the mutant channel. In summary, our data support the concept that altered ion channel properties of PC2 contribute to the pathogenesis of ADPKD.

Novel insights into the modulation of the voltage-gated potassium channel KV1.3 activation gating by membrane ceramides.

Membrane lipids extensively modulate the activation gating of voltage-gated potassium channels (KV), however, much less is known about the mechanisms of ceramide and glucosylceramide actions including which structural element is the main intramolecular target and whether there is any contribution of indirect, membrane biophysics-related mechanisms to their actions. We used two-electrode voltage-clamp fluorometry capable of recording currents and fluorescence signals to simultaneously monitor movements of the pore domain (PD) and the voltage sensor domain (VSD) of the KV1.3 ion channel after attaching an MTS-TAMRA fluorophore to a cysteine introduced into the extracellular S3-S4 loop of the VSD. We observed rightward shifts in the conductance-voltage (G-V) relationship, slower current activation kinetics, and reduced current amplitudes in response to loading the membrane with C16-ceramide (Cer) or C16-glucosylceramide (GlcCer). When analyzing VSD movements, only Cer induced a rightward shift in the fluorescence signal-voltage (F-V) relationship and slowed fluorescence activation kinetics, whereas GlcCer exerted no such effects. These results point at a distinctive mechanism of action with Cer primarily targeting the VSD, while GlcCer only the PD of KV1.3. Using environment-sensitive probes and fluorescence-based approaches, we show that Cer and GlcCer similarly increase molecular order in the inner, hydrophobic regions of bilayers, however, Cer induces a robust molecular reorganization at the membrane-water interface. We propose that this unique ordering effect in the outermost membrane layer in which the main VSD rearrangement involving an outward sliding of the top of S4 occurs can explain the VSD targeting mechanism of Cer, which is unavailable for GlcCer.

Mapping the molecular motions of 5-HT3 serotonin-gated channel by voltage-clamp fluorometry.

The serotonin-gated ion channel (5-HT3R) mediates excitatory neuronal communication in the gut and the brain. It is the target for setrons, a class of competitive antagonists widely used as antiemetics, and is involved in several neurological diseases. Cryo-electron microscopy (cryo-EM) of the 5-HT3R in complex with serotonin or setrons revealed that the protein has access to a wide conformational landscape. However, assigning known high-resolution structures to actual states contributing to the physiological response remains a challenge. In the present study, we used voltage-clamp fluorometry (VCF) to measure simultaneously, for 5-HT3R expressed at a cell membrane, conformational changes by fluorescence and channel opening by electrophysiology. Four positions identified by mutational screening report motions around and outside the serotonin-binding site through incorporation of cysteine-tethered rhodamine dyes with or without a nearby quenching tryptophan. VCF recordings show that the 5-HT3R has access to four families of conformations endowed with distinct fluorescence signatures: 'resting-like' without ligand, 'inhibited-like' with setrons, 'pre-active-like' with partial agonists, and 'active-like' (open channel) with partial and strong agonists. Data are remarkably consistent with cryo-EM structures, the fluorescence partners matching respectively apo, setron-bound, 5-HT bound-closed, and 5-HT-bound-open conformations. Data show that strong agonists promote a concerted motion of all fluorescently labeled sensors during activation, while partial agonists, especially when loss-of-function mutations are engineered, stabilize both active and pre-active conformations. In conclusion, VCF, though the monitoring of electrophysiologically silent conformational changes, illuminates allosteric mechanisms contributing to signal transduction and their differential regulation by important classes of physiological and clinical effectors.

High-Performance Multi-Level Inverter with Symmetry and Simplification.

This paper presents a high-performance, multilevel inverter with symmetry and simplification. This inverter is a single-phase, seven-level symmetric switched-capacitor inverter based on the concept of the double voltage clamping circuit connected to the half-bridge circuit. Above all, only a single DC power supply is used to achieve a single-phase inverter with seven levels and a voltage gain of three. In addition to analyzing the operating principle of the proposed switched-capacitor multilevel inverter in detail, the stability analysis and controller design are carried out by the state-space averaging method. The feasibility and effectiveness of the proposed structure are validated by some simulated results based on the PSIM simulation tool and by some experiments using FPGA as a control kernel, respectively. However, in this study, the switches were implemented by MOSFETs to meet a high switching frequency. These MOSFETs can be replaced by IGBTs in the motor drive applications so that the used switching frequency can be reduced.

Ready-to-Record Cells for Kinetic Screening of VGICs.

Voltage-gated ion channels (VGICs) are integral membrane proteins crucial for transmitting electrical signals in excitable cells. Understanding the kinetics of these ion channels requires conducting patch-clamp experiments using genetically modified cell lines that express a single type of ion channel gene. However, this process relies on the continuous maintenance of cell lines to ensure an adequate supply of sample cells for patch-clamp experiments. Advancements in automated patch-clamp methods have enabled researchers to significantly increase the number of patch-clamped cells per experiment, from just a few cells to as many as 384 cells. Despite this progress, the manual task of preparing the cell samples remains a significant bottleneck in the kinetic screening of VGICs. Here we describe a method to address this challenge by generating ready-to-record (RTR) VGIC-expressing cells that can be frozen and stored separately from patch-clamp experiments. This decoupling of the cell sample preparation process from the patch-clamp experiments offers a streamlined approach to studying VGICs on manual or an automated patch-clamp system.

Voltage Clamp Fluorometry: Illuminating the Dynamics of Ion Channels.

Ion channels comprise one of the largest targets for drug development and treatment and have been a subject of enduring fascination since first discovered in the 1950s. Over the past decades, thousands of publications have explored the cellular biology and molecular physiology of these proteins, and many channel structures have been determined since the late 1990s. Trying to connect the dots between ion channel function and structure, voltage clamp fluorometry (VCF) emerges as a powerful tool because it allows monitoring of the conformational rearrangements underlying the different functional states of the channel. This technique represents an elegant harmonization of molecular biology, electrophysiology, and fluorescence. In the following chapter, we will provide a concise guide to performing VCF on Xenopus laevis oocytes using the two-electrode voltage clamp (TEVC) modality. This is the most widely used configuration on Xenopus oocytes for its relative simplicity and demonstrated success in a number of different ion channels utilizing a variety of attached labels.

Relationship between ion currents and membrane capacitance in canine ventricular myocytes.

Current density, the membrane current value divided by membrane capacitance (Cm), is widely used in cellular electrophysiology. Comparing current densities obtained in different cell populations assume that Cm and ion current magnitudes are linearly related, however data is scarce about this in cardiomyocytes. Therefore, we statistically analyzed the distributions, and the relationship between parameters of canine cardiac ion currents and Cm, and tested if dividing original parameters with Cm had any effect. Under conventional voltage clamp conditions, correlations were high for IK1, moderate for IKr and ICa,L, while negligible for IKs. Correlation between Ito1 peak amplitude and Cm was negligible when analyzing all cells together, however, the analysis showed high correlations when cells of subepicardial, subendocardial or midmyocardial origin were analyzed separately. In action potential voltage clamp experiments IK1, IKr and ICa,L parameters showed high correlations with Cm. For INCX, INa,late and IKs there were low-to-moderate correlations between Cm and these current parameters. Dividing the original current parameters with Cm reduced both the coefficient of variation, and the deviation from normal distribution. The level of correlation between ion currents and Cm varies depending on the ion current studied. This must be considered when evaluating ion current densities in cardiac cells.

The larva and adult of Helicoverpa armigera use differential gustatory receptors to sense sucrose.

Almost all herbivorous insects feed on plants and use sucrose as a feeding stimulant, but the molecular basis of their sucrose reception remains unclear. Helicoverpa armigera as a notorious crop pest worldwide mainly feeds on reproductive organs of many plant species in the larval stage, and its adult draws nectar. In this study, we determined that the sucrose sensory neurons located in the contact chemosensilla on larval maxillary galea were 100-1000 times more sensitive to sucrose than those on adult antennae, tarsi, and proboscis. Using the Xenopus expression system, we discovered that Gr10 highly expressed in the larval sensilla was specifically tuned to sucrose, while Gr6 highly expressed in the adult sensilla responded to fucose, sucrose and fructose. Moreover, using CRISPR/Cas9, we revealed that Gr10 was mainly used by larvae to detect lower sucrose, while Gr6 was primarily used by adults to detect higher sucrose and other saccharides, which results in differences in selectivity and sensitivity between larval and adult sugar sensory neurons. Our results demonstrate the sugar receptors in this moth are evolved to adapt toward the larval and adult foods with different types and amounts of sugar, and fill in a gap in sweet taste of animals.

The Conventional and Breakthrough Tool for the Study of L-Glutamate Transporters.

In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific field, but they are still attractive experimental systems for neuropharmacological studies. We will therefore summarize the pharmacological significance, advantages (especially in the study of EAAT2), and experimental techniques that can be applied to Xenopus oocytes; our new findings concerning L-glutamate (L-Glu) transporters and PUFAs; and the significant outcomes of our data. The data obtained from electrophysiological and molecular biological studies of Xenopus oocytes have provided us with further important questions, such as whether or not some PUFAs can modulate EAATs as allosteric modulators and to what extent docosahexaenoic acid (DHA) affects neurotransmission and thereby affects brain functions. Xenopus oocytes have great advantages in the studies about the interactions between molecules and functional proteins, especially in the case when the expression levels of the proteins are small in cell culture systems without transfections. These are also proper to study the mechanisms underlying the interactions. Based on the data collected in Xenopus oocyte experiments, we can proceed to the next step, i.e., the physiological roles of the compounds and their significances. In the case of EAAT2, the effects on the neurotransmission should be examined by electrophysiological approach using acute brain slices. For new drug development, pharmacokinetics pharmacodynamics (PKPD) data and blood brain barrier (BBB) penetration data are also necessary. In order not to miss the promising candidate compounds at the primary stages of drug development, we should reconsider using Xenopus oocytes in the early phase of drug development.

Electrophysiology of Ctenophore Smooth Muscle.

Unlike in the Cnidaria, where muscle cells are coupled together into an epithelium, ctenophore muscles are single, elongated, intramesogleal structures resembling vertebrate smooth muscle. Under voltage-clamp, these fibers can be separated into different classes with different sets of membrane ion channels. The ion channel makeup is related to the muscle's anatomical position and specific function. For example, Beroe ovata radial fibers, which are responsible for maintaining the rigidity of the body wall, generate sequences of brief action potentials whereas longitudinal fibers, which are concerned with mouth opening and body flexions, often produce single longer duration action potentials.Beroe muscle contractions depend on the influx of Ca2+. During an action potential the inward current is carried by Ca2+, and the increase in intracellular Ca2+ concentration generated can be monitored in FLUO-3-loaded cells. Confocal microscopy in line scan mode shows that the Ca2+ spreads from the outer membrane into the core of the fiber and is cleared from there relatively slowly. The rise in intracellular Ca2+ is linked to an increase in a Ca2+-activated K+ conductance (KCa), which can also be elicited by iontophoretic Ca2+ injection. Near the cell membrane, Ca2+ clearance monitored using FLUO3, matches the decline in the KCa conductance. For light loads, Ca2+ is cleared rapidly, but this fast system is insufficient when Ca2+ influx is maintained. Action potential frequency may be regulated by the slowly developing KCa conductance.

Expression and Functional Analysis of Ctenophore Glutamate Receptor Genes.

The functional analysis of ctenophore neurotransmitter receptors, transporters, and ion channels can be greatly simplified by use of heterologous expression systems. Heterologous expression allows the characterization of individual membrane proteins, expressed at high levels in cells, where background activity by endogenous ion channels and transporters is with few exceptions minimal. The goal of such experiments is to gain an in-depth understanding of the behavior and regulation of individual molecular species, which is challenging in native tissue, but especially so in the case of ctenophores and other marine organisms. Coupled with transcriptome analysis, and immunohistochemical studies of receptor expression in vivo, experiments with heterologous expression systems can provide valuable insight into cellular activity, prior to more challenging functional studies on native tissues.

Loose Coupling between the Voltage Sensor and the Activation Gate in Mammalian HCN Channels Suggests a Gating Mechanism.

Voltage-gated potassium (Kv) channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels share similar structures but have opposite gating polarity. Kv channels have a strong coupling (>109) between the voltage sensor (S4) and the activation gate: when S4s are activated, the gate is open to >80% but, when S4s are deactivated, the gate is open <10-9 of the time. Using noise analysis, we show that the coupling between S4 and the gate is <200 in HCN channels. In addition, using voltage clamp fluorometry, locking the gate open in a Kv channel drastically altered the energetics of S4 movement. In contrast, locking the gate open or decreasing the coupling between S4 and the gate in HCN channels had only minor effects on the energetics of S4 movement, consistent with a weak coupling between S4 and the gate. We propose that this loose coupling is a prerequisite for the reversed voltage gating in HCN channels.

Sugar binding of sodium-glucose cotransporters analyzed by voltage-clamp fluorometry.

Sugar absorption is crucial for life and relies on glucose transporters, including sodium-glucose cotransporters (SGLTs). Although the structure of SGLTs has been resolved, the substrate selectivity of SGLTs across diverse isoforms has not been determined owing to the complex substrate-recognition processes and limited analysis methods. Therefore, this study used voltage-clamp fluorometry (VCF) to explore the substrate-binding affinities of human SGLT1 in Xenopus oocytes. VCF analysis revealed high-affinity binding of D-glucose and D-galactose, which are known transported substrates. D-fructose, which is not a transported substrate, also bound to SGLT1, suggesting potential recognition despite the lack of transport activity. VCF analysis using the T287N mutant of the substrate-binding pocket, which has reduced D-glucose transport capacity, showed that its D-galactose-binding affinity exceeded its D-glucose-binding affinity. This suggests that the change in the VCF signal was due to substrate binding to the binding pocket. Both D-fructose and L-sorbose showed similar binding affinities, indicating that SGLT1 preferentially binds to pyranose-form sugars, including D-fructopyranose. Electrophysiological analysis confirmed that D-fructose binding did not affect the SGLT1 transport function. The significance of the VCF assay lies in its ability to measure sugar-protein interactions in living cells, thereby bridging the gap between structural analyses and functional characterizations of sugar transporters. Our findings also provide insights into SGLT substrate selectivity and the potential for developing medicines with reduced side effects by targeting non-glucose sugars with low bioreactivity.

A synthetic flavonoid derivate in the plasma membrane transforms the voltage-clamp fluorometry signal of CiHv1.

Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.

Selective Potentiation of the (α4)3(ß2)2 Nicotinic Acetylcholine Receptor Response by NS9283 Analogues.

NS9283, 3-(3-pyridyl)-5-(3-cyanophenyl)-1,2,4-oxadiazole, is a selective positive allosteric modulator of (α4)3(ß2)2 nicotinic acetylcholine receptors (nAChRs). It has good subtype selective therapeutic potential afforded by its specific binding to the unique α4-α4 subunit interface present in the (α4)3(ß2)2 nAChR. However, there is currently a lack of structure activity relationship (SAR) studies aimed at developing a class of congeners endowed with the same profile of activity that can help consolidate the druggability of the α4-α4 subunit interface. In this study, new NS9283 analogues were designed, synthesized, and characterized for their ability to selectively potentiate the ACh activity at heterologous (α4)3(ß2)2 nAChRs vs nAChR subtypes (α4)2(ß2)3, α5α4ß2, and α7. With few exceptions, all the NS9283 analogues exerted positive modulation of the (α4)3(ß2)2 nAChR ACh-evoked responses. Above all, those modified at the 3-cyanophenyl moiety by replacement with 3-nitrophenyl (4), 4-cyanophenyl (10), and N-formyl-4-piperidinyl (20) showed the same efficacy as NS9283, although with lower potency. Molecular dynamics simulations of NS9283 and some selected analogues highlighted consistency between potentiation activity and pose of the ligand inside the α4-α4 site with the main interaction being with the complementary (-) side and induction of a significant conformational change of the Trp156 residue in the principal (+) side.

New Alpha9 nAChR Ligands Based on a 5-(Quinuclidin-3-ylmethyl)-1,2,4-oxadiazole Scaffold.

Several lines of evidence have indicated that nicotinic acetylcholine receptors (nAChR) that contain α9 subunits, probably in combination with α10 subunits, may be valuable targets for the management of pain associated with inflammatory diseases through a cholinergic anti-inflammatory system (CAS), which has also been associated with α7 nAChR. Both α7- and α9-containing neuronal nAChR can be pharmacologically distinguished from the high-affinity nicotinic receptors of the brain by their sensitivity to α-bungarotoxin, but in other ways, they have quite distinct pharmacological profiles. The early association of α7 with CAS led to the development of numerous new ligands, variously characterized as α7 agonists, partial agonists, or silent agonists that desensitized α7 receptors without activation. Subsequent reinvestigation of one such family of α7 ligands based on an N,N-diethyl-N'-phenylpiperazine scaffold led to the identification of potent agonists and antagonists for α9. In this paper, we characterize the α9/α10 activity of a series of compounds based on a 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole (QMO) scaffold and identify two new potent ligands of α9, QMO-28, an agonist, and QMO-17, an antagonist. We separated the stereoisomers of these compounds to identify the most potent agonist and discovered that only the 3R isomer of QMO-17 was an α9 antagonist, permitting an in silico model of α9 antagonism to be developed. The α9 activity of these compounds was confirmed to be potentially useful for CAS management of inflammatory pain in cell-based assays of cytokine release.

The small molecule activator S3969 stimulates the epithelial sodium channel by interacting with a specific binding pocket in the channel's ß-subunit.

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969-binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of ß-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics simulations predicted critical residues in the thumb domain of ß-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the ß-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. Molecular dynamics simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of ß-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (ßR437C - γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.