Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes.

The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.

Identification, structural, and biophysical characterization of a positive modulator of human Kv3.1 channels.

Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.

Conformational rearrangements in the second voltage sensor domain switch PIP2- and voltage-gating modes in two-pore channels.

Two-pore channels (TPCs) are activated by phosphatidylinositol bisphosphate (PIP2) binding to domain I and/or by voltage sensing in domain II (DII). Little is known about how these two stimuli are integrated, and how each TPC subtype achieves its unique preference. Here, we show that distinct conformations of DII-S4 in the voltage-sensor domain determine the two gating modes. DII-S4 adopts an intermediate conformation, and forced stabilization in this conformation was found to result in a high PIP2-dependence in primarily voltage-dependent TPC3. In TPC2, which is PIP2-gated and nonvoltage-dependent, a stabilized intermediate conformation does not affect the PIP2-gated currents. These results indicate that the intermediate state represents the PIP2-gating mode, which is distinct from the voltage-gating mode in TPCs. We also found in TPC2 that the tricyclic antidepressant desipramine induces DII-S4-based voltage dependence and that naringenin, a flavonoid, biases the mode preference from PIP2-gating to desipramine-induced voltage gating. Taken together, our study on TPCs revealed an unprecedented mode-switching mechanism involving conformational changes in DII-S4, and its active role in integrating voltage and PIP2 stimuli.

Trp207 regulation of voltage-dependent activation of human Hv1 proton channel.

In voltage-gated Na+ and K+ channels, the hydrophobicity of noncharged residues in the S4 helix has been shown to regulate the S4 movement underlying the process of voltage-sensing domain (VSD) activation. In voltage-gated proton channel Hv1, there is a bulky noncharged tryptophan residue located at the S4 transmembrane segment. This tryptophan remains entirely conserved across all Hv1 members but is not seen in other voltage-gated ion channels, indicating that the tryptophan contributes different roles in VSD activation. The conserved tryptophan of human voltage-gated proton channel Hv1 is Trp207 (W207). Here, we showed that W207 modifies human Hv1 voltage-dependent activation, and small residues replacement at position 207 strongly perturbs Hv1 channel opening and closing, and the size of the side chain instead of the hydrophobic group of W207 regulates the transition between closed and open states of the channel. We conclude that the large side chain of tryptophan controls the energy barrier during the Hv1 VSD transition.

Electrical Impedance Spectroscopy with Bacterial Biofilms: Neuronal-like Behavior.

Negative capacitance at low frequencies for spiking neurons was first demonstrated in 1941 (K. S. Cole) by using extracellular electrodes. The phenomenon subsequently was explained by using the Hodgkin-Huxley model and is due to the activity of voltage-gated potassium ion channels. We show that Escherichia coli (E. coli) biofilms exhibit significant stable negative capacitances at low frequencies when they experience a small DC bias voltage in electrical impedance spectroscopy experiments. Using a frequency domain Hodgkin-Huxley model, we characterize the conditions for the emergence of this feature and demonstrate that the negative capacitance exists only in biofilms containing living cells. Furthermore, we establish the importance of the voltage-gated potassium ion channel, Kch, using knock-down mutants. The experiments provide further evidence for voltage-gated ion channels in E. coli and a new, low-cost method to probe biofilm electrophysiology, e.g., to understand the efficacy of antibiotics. We expect that the majority of bacterial biofilms will demonstrate negative capacitances.

Melatonin alters the excitability of mouse cerebellar granule neurons by inhibiting voltage-gated sodium, potassium, and calcium channels.

Besides its role in the circadian rhythm, the pineal gland hormone melatonin (MLT) also possesses antiepileptogenic, antineoplastic, and cardioprotective properties, among others. The dosages necessary to elicit beneficial effects in these diseases often far surpass physiological concentrations. Although even high doses of MLT are considered to be largely harmless to humans, the possible side effects of pharmacological concentrations are so far not well investigated. In the present study, we report that pharmacological doses of MLT (3 mM) strongly altered the electrophysiological characteristics of cultured primary mouse cerebellar granule cells (CGCs). Using whole-cell patch clamp and ratiometric Ca2+ imaging, we observed that pharmacological concentrations of MLT inhibited several types of voltage-gated Na+ , K+ , and Ca2+ channels in CGCs independently of known MLT-receptors, altering the character and pattern of elicited action potentials (APs) significantly, quickly and reversibly. Specifically, MLT reduced AP frequency, afterhyperpolarization, and rheobase, whereas AP amplitude and threshold potential remained unchanged. The altered biophysical profile of the cells could constitute a possible mechanism underlying the proposed beneficial effects of MLT in brain-related disorders, such as epilepsy. On the other hand, it suggests potential adverse effects of pharmacological MLT concentrations on neurons, which should be considered when using MLT as a pharmacological compound.

Differential Proteomic Analysis of Low-Dose Chronic Paralytic Shellfish Poisoning.

Shellfish poisoning is a common food poisoning. To comprehensively characterize proteome changes in the whole brain due to shellfish poisoning, Tandem mass tag (TMT)-based differential proteomic analysis was performed with a low-dose chronic shellfish poisoning model in mice. A total of 6798 proteins were confidently identified, among which 123 proteins showed significant changes (fold changes of >1.2 or <0.83, p < 0.05). In positive regulation of synaptic transmission, proteins assigned to a presynaptic membrane (e.g., Grik2) and synaptic transmission (e.g., Fmr1) changed. In addition, altered proteins in nervous system development were observed, suggesting that mice suffered nerve damage due to the nervous system being activated. Ion transport in model mice was demonstrated by a decrease in key enzymes (e.g., Kcnj11) in voltage-gated ion channel activity and solute carrier family (e.g., Slc38a3). Meanwhile, alterations in transferase activity proteins were observed. In conclusion, these modifications observed in brain proteins between the model and control mice provide valuable insights into understanding the functional mechanisms underlying shellfish poisoning.

The Phytochemical, Quercetin, Attenuates Nociceptive and Pathological Pain: Neurophysiological Mechanisms and Therapeutic Potential.

Although phytochemicals are plant-derived toxins that are primarily produced as a form of defense against insects or microbes, several lines of study have demonstrated that the phytochemical, quercetin, has several beneficial biological actions for human health, including antioxidant and inflammatory effects without side effects. Quercetin is a flavonoid that is widely found in fruits and vegetables. Since recent studies have demonstrated that quercetin can modulate neuronal excitability in the nervous system, including nociceptive sensory transmission via mechanoreceptors and voltage-gated ion channels, and inhibit the cyclooxygenase-2-cascade, it is possible that quercetin could be a complementary alternative medicine candidate; specifically, a therapeutic agent against nociceptive and pathological pain. The focus of this review is to elucidate the neurophysiological mechanisms underlying the modulatory effects of quercetin on nociceptive neuronal activity under nociceptive and pathological conditions, without inducing side effects. Based on the results of our previous research on trigeminal pain, we have confirmed in vivo that the phytochemical, quercetin, demonstrates (i) a local anesthetic effect on nociceptive pain, (ii) a local anesthetic effect on pain related to acute inflammation, and (iii) an anti-inflammatory effect on chronic pain. In addition, we discuss the contribution of quercetin to the relief of nociceptive and inflammatory pain and its potential clinical application.

Reduced Graphene Oxides Modulate the Expression of Cell Receptors and Voltage-Dependent Ion Channel Genes of Glioblastoma Multiforme.

The development of nanotechnology based on graphene and its derivatives has aroused great scientific interest because of their unusual properties. Graphene (GN) and its derivatives, such as reduced graphene oxide (rGO), exhibit antitumor effects on glioblastoma multiforme (GBM) cells in vitro. The antitumor activity of rGO with different contents of oxygen-containing functional groups and GN was compared. Using FTIR (fourier transform infrared) analysis, the content of individual functional groups (GN/exfoliation (ExF), rGO/thermal (Term), rGO/ammonium thiosulphate (ATS), and rGO/ thiourea dioxide (TUD)) was determined. Cell membrane damage, as well as changes in the cell membrane potential, was analyzed. Additionally, the gene expression of voltage-dependent ion channels (clcn3, clcn6, cacna1b, cacna1d, nalcn, kcne4, kcnj10, and kcnb1) and extracellular receptors was determined. A reduction in the potential of the U87 glioma cell membrane was observed after treatment with rGO/ATS and rGO/TUD flakes. Moreover, it was also demonstrated that major changes in the expression of voltage-dependent ion channel genes were observed in clcn3, nalcn, and kcne4 after treatment with rGO/ATS and rGO/TUD flakes. Furthermore, the GN/ExF, rGO/ATS, and rGO/TUD flakes significantly reduced the expression of extracellular receptors (uPar, CD105) in U87 glioblastoma cells. In conclusion, the cytotoxic mechanism of rGO flakes may depend on the presence and types of oxygen-containing functional groups, which are more abundant in rGO compared to GN.

Functional characterization and optimization of a bacterial cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels produce the initial electrical signal in mammalian vision and olfaction. They open in response to direct binding of cyclic nucleotide (cAMP or cGMP) to a cytoplasmic region of the channel. However, the conformational rearrangements occurring upon binding to produce pore opening (i.e. gating) are not well understood. SthK is a bacterial CNG channel that has the potential to serve as an ideal model for structure-function studies of gating but is currently limited by its toxicity, native cysteines, and low open probability (Po). Here, we expressed SthK in giant Escherichia coli spheroplasts and performed patch-clamp recordings to characterize SthK gating in a bacterial membrane. We demonstrated that the Po in cAMP is higher than has been previously published and that cGMP acts as a weak partial SthK agonist. Additionally, we determined that SthK expression is toxic to E. coli because of gating by cytoplasmic cAMP. We overcame this toxicity by developing an adenylate cyclase-knockout E. coli cell line. Finally, we generated a cysteine-free SthK construct and introduced mutations that further increase the Po in cAMP. We propose that this SthK model will help elucidate the gating mechanism of CNG channels.

High-Throughput Fluorescence Assays for Ion Channels and GPCRs.

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs.

Analysis of Single-Nucleotide Polymorphisms in Human Voltage-Gated Ion Channels.

Voltage-gated ion channels (VGICs) are one of the largest groups of transmembrane proteins. Due to their major role in the generation and propagation of electrical signals, VGICs are considered important from a medical viewpoint, and their dysfunction is often associated with Channelopathies. We identified disease-associated mutations and polymorphisms in these proteins through mapping missense single-nucleotide polymorphisms from the UniProt and ClinVar databases on their amino acid sequence, considering their special topological and functional characteristics. Statistical analysis revealed that disease-associated SNPs are mostly found in the voltage sensor domain and the pore loop. Both of these regions are extremely important for the activation and ion conductivity of VGICs. Moreover, among the most frequently observed mutations are those of arginine to glutamine, to histidine or to cysteine, which can probably be attributed to the extremely important role of arginine residues in the regulation of membrane potential in these proteins. We suggest that topological information in combination with genetic variation data can contribute toward a better evaluation of the effect of currently unclassified mutations in VGICs. It is hoped that potential associations with certain disease phenotypes will be revealed in the future with the use of similar approaches.

Challenges and advances in atomistic simulations of potassium and sodium ion channel gating and permeation.

Ion channels are implicated in many essential physiological events such as electrical signal propagation and cellular communication. The advent of K+ and Na+ ion channel structure determination has facilitated numerous investigations of molecular determinants of their behaviour. At the same time, rapid development of computer hardware and molecular simulation methodologies has made computational studies of large biological molecules in all-atom representation tractable. The concurrent evolution of experimental structural biology with biomolecular computer modelling has yielded mechanistic details of fundamental processes unavailable through experiments alone, such as ion conduction and ion channel gating. This review is a short survey of the atomistic computational investigations of K+ and Na+ ion channels, focusing on KcsA and several voltage-gated channels from the KV and NaV families, which have garnered many successes and engendered several long-standing controversies regarding the nature of their structure-function relationship. We review the latest advancements and challenges facing the field of molecular modelling and simulation regarding the structural and energetic determinants of ion channel function and their agreement with experimental observations.

Nociceptor Signalling through ion Channel Regulation via GPCRs.

The prime task of nociceptors is the transformation of noxious stimuli into action potentials that are propagated along the neurites of nociceptive neurons from the periphery to the spinal cord. This function of nociceptors relies on the coordinated operation of a variety of ion channels. In this review, we summarize how members of nine different families of ion channels expressed in sensory neurons contribute to nociception. Furthermore, data on 35 different types of G protein coupled receptors are presented, activation of which controls the gating of the aforementioned ion channels. These receptors are not only targeted by more than 20 separate endogenous modulators, but can also be affected by pharmacotherapeutic agents. Thereby, this review provides information on how ion channel modulation via G protein coupled receptors in nociceptors can be exploited to provide improved analgesic therapy.

Induction of divalent cation permeability by heterologous expression of a voltage sensor domain.

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K+ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7.

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNaV1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNaV1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNaV1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNaV1.7 channel inhibitor.

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating.

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (µ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.

Action potentials and ion conductances in wild-type and CALHM1-knockout type II taste cells.

Taste bud type II cells fire action potentials in response to tastants, triggering nonvesicular ATP release to gustatory neurons via voltage-gated CALHM1-associated ion channels. Whereas CALHM1 regulates mouse cortical neuron excitability, its roles in regulating type II cell excitability are unknown. In this study, we compared membrane conductances and action potentials in single identified TRPM5-GFP-expressing circumvallate papillae type II cells acutely isolated from wild-type (WT) and Calhm1 knockout (KO) mice. The activation kinetics of large voltage-gated outward currents were accelerated in cells from Calhm1 KO mice, and their associated nonselective tail currents, previously shown to be highly correlated with ATP release, were completely absent in Calhm1 KO cells, suggesting that CALHM1 contributes to all of these currents. Calhm1 deletion did not significantly alter resting membrane potential or input resistance, the amplitudes and kinetics of Na+ currents either estimated from action potentials or recorded from steady-state voltage pulses, or action potential threshold, overshoot peak, afterhyperpolarization, and firing frequency. However, Calhm1 deletion reduced the half-widths of action potentials and accelerated the deactivation kinetics of transient outward currents, suggesting that the CALHM1-associated conductance becomes activated during the repolarization phase of action potentials.NEW & NOTEWORTHY CALHM1 is an essential ion channel component of the ATP neurotransmitter release mechanism in type II taste bud cells. Its contribution to type II cell resting membrane properties and excitability is unknown. Nonselective voltage-gated currents, previously associated with ATP release, were absent in cells lacking CALHM1. Calhm1 deletion was without effects on resting membrane properties or voltage-gated Na+ and K+ channels but contributed modestly to the kinetics of action potentials.

Contributions of the membrane dipole potential to the function of voltage-gated cation channels and modulation by small molecule potentiators.

The membrane dipole potential (Ψd) constitutes one of three electrical potentials generated by cell membranes. Ψd arises from the unfavorable parallel alignment of phospholipid and water dipoles, and varies in magnitude both longitudinally and laterally across the bilayer according to membrane composition and phospholipid packing density. In this work, we propose that dynamic counter-balancing between Ψd and the transmembrane potential (ΔΨm) governs the conformational state transitions of voltage-gated ion channels. Ψd consists of 1) static outer, and dynamic inner leaflet components (Ψd(extra) and Ψd(intra), respectively); and 2) a transmembrane component (ΔΨd(inner-outer)), ariing from differences in intra- and extracellular leaflet composition. Ψd(intra), which transitions between high and low energy states (Ψd(intra, high) and Ψd(intra, low)) as a function of channel conformation, is transduced by the pore domain. ΔΨd(inner-outer) is transduced by the voltage-sensing (VS) domain in summation with ΔΨm. Potentiation of voltage-gated ion channels is of interest for the treatment of cardiac, neuronal, and other disorders arising from inherited/acquired ion channel dysfunction. Potentiators are widely believed to alter the rates and voltage-dependencies of channel gating transitions by binding to pockets in the membrane-facing and other regions of ion channel targets. Here, we propose that potentiators alter Ψd(intra) and/or Ψd(extra), thereby increasing or decreasing the energy barriers governing channel gating transitions. We used quantum mechanical and molecular dynamics (MD) simulations to predict the overall Ψd-modulating effects of a series of published positive hERG potentiators partitioned into model DOPC bilayers. Our findings suggest a strong correlation between the magnitude of Ψd-lowering and positive hERG potentiation across the series.

Modulation of voltage-gated conductances of retinal horizontal cells by UV-excited TiO2 nanoparticles.

This study examines the ability of optically-excited titanium dioxide nanoparticles to influence voltage-gated ion channels in retinal horizontal cells. Voltage clamp recordings were obtained in the presence and absence of TiO2 and ultraviolet laser excitation. Significant current changes were observed in response to UV light, particularly in the -40 mV to +40 mV region where voltage-gated Na+ and K+ channels have the highest conductance. Cells in proximity to UV-excited TiO2 exhibited a left-shift in the current-voltage relation of around 10 mV in the activation of Na+ currents. These trends were not observed in control experiments where cells were excited with UV light without being exposed to TiO2. Electrostatic force microscopy confirmed that electric fields can be induced in TiO2 with UV light. Simulations using the Hodgkin-Huxley model yielded results which agreed with the experimental data and showed the I-V characteristics of individual ion channels in the presence of UV-excited TiO2.