Voltage-sensing Domain Research Articles

PUFA stabilizes a conductive state of the selectivity filter in IKs channels.

In cardiomyocytes, the KCNQ1/KCNE1 channel complex mediates the slow delayed-rectifier current (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause long QT syndrome (LQTS), a syndrome with a prolonged QT interval on the ECG, which increases the risk of ventricular arrhythmia and sudden cardiac death. One potential therapeutical intervention for LQTS is based on targeting IKs channels to restore channel function and/or the physiological QT interval. Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels and activate IKs channels by binding to two different sites, one in the voltage sensor domain - which shifts the voltage dependence to more negative voltages - and the other in the pore domain - which increases the maximal conductance of the channels (Gmax). However, the mechanism by which PUFAs increase the Gmax of the IKs channels is still poorly understood. In addition, it is unclear why IKs channels have a very small single-channel conductance and a low open probability or whether PUFAs affect any of these properties of IKs channels. Our results suggest that the selectivity filter in KCNQ1 is normally unstable, contributing to the low open probability, and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state.

PUFA stabilizes a conductive state of the selectivity filter in IKs channels

In cardiomyocytes, the KCNQ1/KCNE1 channel complex mediates the slow delayed-rectifier current (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause long QT syndrome (LQTS), a syndrome with a prolonged QT interval on the ECG, which increases the risk of ventricular arrhythmia and sudden cardiac death. One potential therapeutical intervention for LQTS is based on targeting IKs channels to restore channel function and/or the physiological QT interval. Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels and activate IKs channels by binding to two different sites, one in the voltage sensor domain – which shifts the voltage dependence to more negative voltages – and the other in the pore domain – which increases the maximal conductance of the channels (Gmax). However, the mechanism by which PUFAs increase the Gmax of the IKs channels is still poorly understood. In addition, it is unclear why IKs channels have a very small single-channel conductance and a low open probability or whether PUFAs affect any of these properties of IKs channels. Our results suggest that the selectivity filter in KCNQ1 is normally unstable, contributing to the low open probability, and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state.

Functional expression of the chimera proteins of Nav1.7 and NavAb in Escherichiacoli

Nav1.7 is a eukaryotic voltage-dependent Na channel (Nav) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I–IV). It is involved in pain perception, and VSDs that differ significantly by Nav subtype are targeted in the development of Nav1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Nav1.7 inhibitors, the chimera protein (NavAb/1.7VSD) of Nav from prokaryotic Arcobacter butzleri (NavAb), in which extracellular loops of VSD were replaced with homologous loops from Nav1.7, serves as an effective model. This is because NavAb harbors only one VSD and the biological activity of NavAb/1.7VSD was previously confirmed. To date, NavAb/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of NavAb/1.7VSD-II in Escherichia coli (E. coli). The expression of this protein in the inner membrane of E. coli was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Nav1.7-specific peptide inhibitor. The results indicate that NavAb/1.7VSD-II was functionally expressed in E. coli, providing empirical support for the discovery of new VSD-specific Nav1.7 inhibitors using the PERISS screening method.

Structural Basis for Voltage Gating and Dalfampridine Binding in the Shaker Potassium Channel.

The generation and propagation of action potentials in neurons depend on the coordinated activation of voltage-dependent sodium and potassium channels. Potassium channels of the Shaker family regulate neuronal excitability through voltage-dependent opening and closing of their ion conduction pore. This family of channels is an important therapeutic target, particularly in multiple sclerosis where the inhibitor dalfampridine (4-aminopyridine) is used to improve nerve conduction. The molecular details of how the voltage sensor domain drives opening of the pore domain has been limited by the lack of closed-state structures, also impairing the search for novel drugs. Using AlphaFold2-based conformational sampling methods we identify a structural model for the closed Shaker potassium channel where movement of the voltage sensor drives the opening trough interactions between the S4-S5 linker and S6 helix. We show experimentally that breakage of a backbone hydrogen bond is a critical part of the activation pathway. Through docking we identify a hydrophobic cavity formed by the pore domain helices that binds dalfampridine in the closed state. Our results demonstrate how voltage sensor movement drives pore opening and provide a structural framework for developing new therapeutic agents targeting the closed state. We anticipate this work will enable structure-based drug design efforts focused on state-dependent modulation of voltage-gated ion channels for the treatment of neurological disorders.

Short Lysine-Containing Tripeptide as Analgesic Substance: The Possible Mechanism of Ligand–Receptor Binding to the Slow Sodium Channel

A possible molecular mechanism of the ligand–receptor binding of Ac-Lys-Lys-Lys-NH2 (Ac-KKK-NH2) to the NaV1.8 channel that is responsible for nociceptive signal coding in the peripheral nervous system is investigated by a number of experimental and theoretical techniques. Upon Ac-KKK-NH2 application at 100 nM, a significant decrease in the effective charge carried by the NaV1.8 channel activation gating system Zeff is demonstrated in the patch-clamp experiments. A strong Ac-KKK-NH2 analgesic effect at both the spinal and supraspinal levels is detected in vivo in the formalin test. The distances between the positively charged amino groups in the Ac-KKK-NH2 molecule upon binding to the NaV1.8 channel are 11–12 Å, as revealed by the conformational analysis. The blind docking with the NaV1.8 channel has made it possible to locate the Ac-KKK-NH2 binding site on the extracellular side of the voltage-sensing domain VSDI. The Ac-KKK-NH2 amino groups are shown to form ionic bonds with Asp151 and Glu157 and a hydrogen bond with Thr161, which affects the coordinated movement of the voltage sensor up and down, thus modulating the Zeff value. According to the results presented, Ac-KKK-NH2 is a promising candidate for the role of an analgesic medicinal substance that can be applied for pain relief in humans.

ω-Grammotoxin-SIA inhibits voltage-gated Na+ channel currents.

ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide.

Intra-channel bi-epitopic crosslinking unleashes ultrapotent antibodies targeting NaV1.7 for pain alleviation.

Crucial for cell activities, ion channels are key drug discovery targets. Although small-molecule and peptide modulators dominate ion channel drug discovery, antibodies are emerging as an alternative modality. However, challenges persist in generating potent antibodies, especially for channels with limited extracellular epitopes. We herein present a bi-epitopic crosslinking strategy to overcome these challenges, focusing on NaV1.7, a potential analgesic target. Aiming to crosslink two non-overlapping epitopes on voltage-sensing domains II and IV, we construct bispecific antibodies and ligand-antibody conjugates. Enhanced affinity and potency are observed in comparison to the monospecific controls. Among them, a ligand-antibody conjugate (1080-PEG7-ACDTB) displays a two-orders-of-magnitude improvement in potency (IC50 of 0.06± 0.01nM) and over 1,000-fold selectivity for NaV1.7. Additionally, this conjugate demonstrates robust analgesic effects in mouse pain models. Our study introduces an approach to developing effective antibodies against NaV1.7, thereby initiating a promising direction for the advancement of pain therapeutics.

A Rich Conformational Palette Underlies Human CaV2.1-Channel Availability.

Depolarization-evoked opening of CaV2.1 (P/Q-type) Ca2+-channels triggers neurotransmitter release, while voltage-dependent inactivation (VDI) limits channel availability to open, contributing to synaptic plasticity. The mechanism of CaV2.1 response to voltage is unclear. Using voltage-clamp fluorometry and kinetic modeling, we optically tracked and physically characterized the structural dynamics of the four CaV2.1 voltage-sensor domains (VSDs). VSD-I seems to directly drive opening and convert between two modes of function, associated with VDI. VSD-II is apparently voltage-insensitive. VSD-III and VSD-IV sense more negative voltages and undergo voltage-dependent conversion uncorrelated with VDI. Auxiliary -subunits regulate VSD-I-to-pore coupling and VSD conversion kinetics. CaV2.1 VSDs are differentially sensitive to voltage changes brief and long-lived. Specifically the voltage-dependent conformational changes of VSD-I are linked to synaptic release and plasticity.

PIP2 inhibits pore opening of the cyclic nucleotide-gated channel SthK

The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels. The prokaryotic SthK channel from Spirochaeta thermophila shares features with CNG and HCN channels and is an established model for this channel family. Here, we show SthK activity is inhibited by PIP2. A cryo-EM structure of SthK in nanodiscs reveals a PIP2-fitting density coordinated by arginine and lysine residues from the S4 helix and the C-linker, located between voltage-sensing and pore domains of adjacent subunits. Mutation of two arginine residues weakens PIP2 inhibition with the double mutant displaying insensitivity to PIP2. We propose that PIP2 inhibits SthK by gluing S4 and S6 together, stabilizing a resting channel conformation. The PIP2 binding site is partially conserved in CNG channels suggesting the possibility of a similar inhibition mechanism in the eukaryotic homologs.

Lipophilic compounds restore function to neurodevelopmental-associated KCNQ3 mutations

A major driver of neuronal hyperexcitability is dysfunction of K+ channels, including voltage-gated KCNQ2/3 channels. Their hyperpolarized midpoint of activation and slow activation and deactivation kinetics produce a current that regulates membrane potential and impedes repetitive firing. Inherited mutations in KCNQ2 and KCNQ3 are linked to a wide spectrum of neurodevelopmental disorders (NDDs), ranging from benign familial neonatal seizures to severe epileptic encephalopathies and autism spectrum disorders. However, the impact of these variants on the molecular mechanisms underlying KCNQ3 channel function remains poorly understood and existing treatments have significant side effects. Here, we use voltage clamp fluorometry, molecular dynamic simulations, and electrophysiology to investigate NDD-associated variants in KCNQ3 channels. We identified two distinctive mechanisms by which loss– and gain–of function NDD-associated mutations in KCNQ3 affect channel gating: one directly affects S4 movement while the other changes S4-to-pore coupling. MD simulations and electrophysiology revealed that polyunsaturated fatty acids (PUFAs) primarily target the voltage-sensing domain in its activated conformation and form a weaker interaction with the channel’s pore. Consistently, two such compounds yielded partial and complete functional restoration in R227Q- and R236C-containing channels, respectively. Our results reveal the potential of PUFAs to be developed into therapies for diverse KCNQ3-based channelopathies.

Voltage-dependent G-protein regulation of CaV2.2 (N-type) channels.

How G proteins inhibit N-type, voltage-gated, calcium-selective channels (CaV2.2) during presynaptic inhibition is a decades-old question. G proteins Gβγ bind to intracellular CaV2.2 regions, but the inhibition is voltage dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gβγ acts by selectively inhibiting a subset of the four different CaV2.2 voltage-sensor domains (VSDs I to IV). During regular "willing" gating, VSD-I and -IV activations resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gβγ, CaV2.2 gating is "reluctant": pore opening and VSD I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited, while VSD III is not. We propose that Gβγ inhibition of VSDs I and IV underlies reluctant CaV2.2 gating and subsequent presynaptic inhibition.

A Deep Learning Approach to Uncover Voltage-Gated Ion Channels' Intermediate States.

Owing to recent advancements in cryo-electron microscopy, voltage-gated ion channels have gained a greater comprehension of their structural characteristics. However, a significant enigma remains unsolved for a large majority of these channels: their gating mechanism. This mechanism, which encompasses the conformational changes between open and closed states, is pivotal to their proper functioning. Beyond the binary states of open and closed, an ensemble of intermediate states defines the transition path in-between. Due to the lack of experimental data, one might resort to molecular dynamics simulations as an alternative to decipher these states and the transitions between them. However, the high-energy barriers and the colossal time scales involved hinder access to the latter. We present here an application of deep learning as a reliable pipeline for a comprehensive exploration of voltage-gated ion channel conformational rearrangements during gating. We showcase the pipeline performance specifically on the Kv1.2 voltage sensor domain and confront the results with existing data. We demonstrate how our physics-based deep learning approach contributes to the theoretical understanding of these channels and how it might provide further insights into the exploration of channelopathies.

ELUCIDATING THE DIFFERENTIAL IMPACTS OF EQUIVALENT GATING-CHARGE MUTATIONS IN VOLTAGE-GATED SODIUM CHANNELS.

Voltage-gated sodium (Nav) channels are pivotal for cellular signaling and mutations in Nav channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSDI and VSDII of the cardiac sodium channel Nav1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 μs molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our μs-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure-function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.

A venom peptide-induced NaV channel modulation mechanism involving the interplay between fixed channel charges and ionic gradients

Venoms are used by arthropods either to immobilise prey or as defence against predators. Our study focuses on the venom peptide, Ta3a, from the African ant species, Tetramorium africanum and its effects on voltage-gated sodium (NaV) channels, which are ion channels responsible for the generation of electrical signals in electrically excitable cells, such as neurons. Using the NaV1.7 isoform as our model NaV channel we show that Ta3a prolongs single channel active periods with increased open probability and induces non-inactivating whole-cell currents. Ta3a-affected NaV1.7 channels exhibit a leftward (hyperpolarising) shift in activation threshold, constitutive activity even in the absence of an activating voltage stimulus, and at cell membrane voltages where channels are normally silent. Current-voltage experiments show that Ta3a shifts the voltage at which NaV current changes direction (reversal potential) by altering the local ionic concentration of permeant ions (Na+) rather than changing the channel’s preference for ionic species. We propose a model where Ta3a maintains the positively charged voltage-sensing (S4) domains of the channel in the activated configuration where their electric field is exposed to the extracellular membrane surface to create an ionic bilayer comprising S4 domains and mobile anions (Cl−). This bilayer has a depolarising effect on the cell membrane, thus reducing the amount of externally applied voltage required for channel activation.

CaV1.1 voltage-sensing domain III exclusively controls skeletal muscle excitation-contraction coupling

Skeletal muscle contractions are initiated by action potentials, which are sensed by the voltage-gated calcium channel (CaV1.1) and are conformationally coupled to calcium release from intracellular stores. Notably, CaV1.1 contains four separate voltage-sensing domains (VSDs), which activate channel gating and excitation-contraction (EC-) coupling at different voltages and with distinct kinetics. Here we show that a single VSD of CaV1.1 controls skeletal muscle EC-coupling. Whereas mutations in VSDs I, II and IV affect the current properties but not EC-coupling, only mutations in VSD III alter the voltage-dependence of depolarization-induced calcium release. Molecular dynamics simulations reveal comprehensive, non-canonical state transitions of VSD III in response to membrane depolarization. Identifying the voltage sensor that activates EC-coupling and detecting its unique conformational changes opens the door to unraveling the downstream events linking VSD III motion to the opening of the calcium release channel, and thus resolving the signal transduction mechanism of skeletal muscle EC-coupling.

Harnessing Deep Learning Methods for Voltage-Gated Ion Channel Drug Discovery.

Voltage-gated ion channels (VGICs) are pivotal in regulating electrical activity in excitable cells and are critical pharmaceutical targets for treating many diseases including cardiac arrhythmia and neuropathic pain. Despite their significance, challenges such as achieving target selectivity persist in VGIC drug development. Recent progress in deep learning, particularly diffusion models, has enabled the computational design of protein binders for any clinically relevant protein based solely on its structure. These developments coincide with a surge in experimental structural data for VGICs, providing a rich foundation for computational design efforts. This review explores the recent advancements in computational protein design using deep learning and diffusion methods, focusing on their application in designing protein binders to modulate VGIC activity. We discuss the potential use of these methods to computationally design protein binders targeting different regions of VGICs, including the pore domain, voltage-sensing domains, and interface with auxiliary subunits. We provide a comprehensive overview of the different design scenarios, discuss key structural considerations, and address the practical challenges in developing VGIC-targeting protein binders. By exploring these innovative computational methods, we aim to provide a framework for developing novel strategies that could significantly advance VGIC pharmacology and lead to the discovery of effective and safe therapeutics.

Scorpion α-toxin LqhαIT specifically interacts with a glycan at the pore domain of voltage-gated sodium channels

Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on Nav channels. To elucidate the mechanism of action of LqhαIT, we determined a 3.9Å cryoelectron microscopy (cryo-EM) structure of LqhαIT in complex with the Nav channel from Periplaneta americana (NavPas). We found that LqhαIT binds to voltage sensor domain 4 and traps it in an "S4 down" conformation. The functionally essential C-terminal epitope of LqhαIT forms an extensive interface with the glycan scaffold linked to Asn330 of NavPas that augments a small protein-protein interface between NavPas and LqhαIT. A combination of molecular dynamics simulations, structural comparisons, and prior mutagenesis experiments demonstrates the functional importance of this toxin-glycan interaction. These findings establish a structural basis for the specificity achieved by scorpion α-toxins and reveal the conserved glycan as an essential component of the toxin-binding epitope.

Turning a Kv channel into hot and cold receptor by perturbing its electromechanical coupling.

Voltage-dependent potassium channels (Kv) are extremely sensitive to membrane voltage and play a crucial role in membrane repolarization during action potentials. Kv channels undergo voltage-dependent transitions between closed states before opening. Despite all we have learned using electrophysiological methods and structural studies, we still lack a detailed picture of the energetics of the activation process. We show here that even a single mutation can drastically modify the temperature response of the Shaker Kv channel. Using rapid cell membrane temperature steps (Tsteps), we explored the effects of temperature on the ILT mutant (V369I, I372L, and S376T) and the I384N mutant. The ILT mutant produces a significant separation between the transitions of the voltage sensor domain (VSD) activation and the I384N uncouples its movement from the opening of the domain (PD). ILT and I384N respond to temperature in drastically different ways. In ILT, temperature facilitates the opening of the channel akin to a "hot" receptor, reflecting the temperature dependence of the voltage sensor's last transition and facilitating VSD to PD coupling (electromechanical coupling). In I384N, temperature stabilizes the channel closed configuration analogous to a "cold" receptor. Since I384N drastically uncouples the VSD from the pore opening, we reveal the intrinsic temperature dependence of the PD itself. Here, we propose that the electromechanical coupling has either a "loose" or "tight" conformation. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to the S6 gate. In the tight conformation the VSD activation is more effectively translated into the opening of the PD. This conformational switch can be tuned by temperature and modifications of the S4 and S4-S5 linker. Our results show that we can modulate the temperature dependence of Kv channels by affecting its electromechanical coupling.

Descending to inhibit: Antagonist-induced downward shift of VSD II in TPC2

In this issue of Structure, Chi etal.1 report structural and functional studies that reveal the inhibition mechanism of the lysosomal two-pore channel TPC2 by the antagonist SG-094, which is of interest for drug development. Antagonist binding induces the downward displacement of the voltage-sensor domain II (VSD II), which is accompanied by asymmetric conformational rearrangements of the entire channel.

Different fluorescent labels report distinct components of spHCN channel voltage sensor movement.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.