Voltage-gated Proton Channel Research Articles

Zn2+ to probe voltage-gated proton (Hv1) channels.

Cherny and coworkers use zinc ion as a probe to identify different conformational states of voltage-gated proton (Hv1) channels.

Engineered high-affinity zinc binding site reveals gating configurations of a human proton channel.

The voltage-gated proton channel (HV1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hHV1, current was potently inhibited by externally applied Zn2+ in a construct lacking the two His that bind Zn2+ in WT channels. High-affinity binding with profound effects at 10 nM Zn2+ at pHo 7 suggests additional groups contribute. We hypothesized that Asp185, which faces position 116 in our closed-state model, contributes to Zn2+ chelation. Confirming this prediction, V116H/D185N abolished Zn2+ binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn2+ slowed activation, producing a time constant independent of both voltage and Zn2+ concentration. We hypothesized that slow turn-on of H+ current in the presence of Zn2+ reflects the rate of Zn2+ unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn2+ is greater in the closed state of hHV1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn2+. The lower affinity of Zn2+ in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg205 near position 116, electrostatically expelling Zn2+. This phenomenon provides direct evidence that Asp185 opposes position 116 in closed channels and that Arg205 moves between them when the channel opens.

Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury

BackgroundSpinal cord injury (SCI) causes neurological dysfunction with devastating consequences. SCI pathogenesis is accompanied by inflammasome activation and neuronal damage. But the spatial pattern and the time course of neuronal pyroptosis and apoptosis after SCI should be further elucidated. The microglial voltage-gated proton channel (Hv1) is implicated in reactive oxygen species (ROS)-induced neuronal damage following ischemic stroke. However, there is a lack of quantification on the neuronal pyroptosis and apoptosis associated with microglial Hv1 after SCI.MethodsWe analyzed spatial and temporal characteristics of neuronal pyroptosis and apoptosis following SCI and investigated the effects of Hv1 deficiency on neuronal pyroptosis and the nod-like receptor 3 (NLRP3) inflammasome pathway by using a mouse model of SCI. We tested the effects of Hv1-deficient microglia on ROS production in vivo and examined the relationship between ROS and neuronal pyroptosis in vitro.ResultsWe observed that apoptosis was detected closer to the injury core than pyroptosis. The incidence of neuronal apoptosis peaked on day 1 after SCI and occurred before pyroptosis. Hv1 deficiency reduced neuronal apoptosis and NLRP3-inflammasome-mediated pyroptosis, improved axonal regeneration, and reduced motor deficits. SCI led to elevated ROS levels, whereas Hv1 deficiency downregulated microglial ROS generation. In vitro, ROS upregulated neuronal pyroptosis and activated the NLRP3 inflammasome pathway, both of which were reversed by addition of a ROS scavenger. Our results suggested that microglial Hv1 regulated neuronal apoptosis and NLRP3-induced neuronal pyroptosis after SCI by mediating ROS production.ConclusionFollowing SCI, neuronal pyroptosis lasted longer and occurred farther away from the injury core compared with that of neuronal apoptosis. Microglial Hv1 deficiency downregulated microglial ROS generation and reduced apoptosis and NLRP3-induced neuronal pyroptosis. Our findings may provide novel insights into Hv1-associated mechanisms underlying neuronal damage after SCI.

Insights on small molecule binding to the Hv1 proton channel from free energy calculations with molecular dynamics simulations

Hv1 is a voltage-gated proton channel whose main function is to facilitate extrusion of protons from the cell. The development of effective channel blockers for Hv1 can lead to new therapeutics for the treatment of maladies related to Hv1 dysfunction. Although the mechanism of proton permeation in Hv1 remains to be elucidated, a series of small molecules have been discovered to inhibit Hv1. Here, we computed relative binding free energies of a prototypical Hv1 blocker on a model of human Hv1 in an open state. We used alchemical free energy perturbation techniques based on atomistic molecular dynamics simulations. The results support our proposed open state model and shed light on the preferred tautomeric state of the channel blocker. This work lays the groundwork for future studies on adapting the blocker molecule for more effective inhibition of Hv1.

Dimer interaction in the Hv1 proton channel

The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel superfamily, which stands out in design: It is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4), and a gate (S1) and forming its own ion channel. Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s. Interactions between the transmembrane portions of the subunits may also contribute, but the nature of transmembrane packing is unclear. Using functional analysis of a mutagenesis scan, biochemistry, and modeling, we find that the subunits form a dimer interface along the entire length of S1, and also have intersubunit contacts between S1 and S4. These interactions exert a strong effect on gating, in particular on the stability of the open state. Our results suggest that gating in Hv1 is tuned by extensive VSD-VSD interactions between the gates and voltage sensors of the dimeric channel.

Correction: Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells.

[This corrects the article DOI: 10.1371/journal.pone.0227522.].

Voltage-dependent structural models of the human Hv1 proton channel from long-timescale molecular dynamics simulations

The voltage-gated Hv1 proton channel is a ubiquitous membrane protein that has roles in a variety of cellular processes, including proton extrusion, pH regulation, production of reactive oxygen species, proliferation of cancer cells, and increased brain damage during ischemic stroke. A crystal structure of an Hv1 construct in a putative closed state has been reported, and structural models for the channel open state have been proposed, but a complete characterization of the Hv1 conformational dynamics under an applied membrane potential has been elusive. We report structural models of the Hv1 voltage-sensing domain (VSD), both in a hyperpolarized state and a depolarized state resulting from voltage-dependent conformational changes during a 10-μs-timescale atomistic molecular dynamics simulation in an explicit membrane environment. In response to a depolarizing membrane potential, the S4 helix undergoes an outward displacement, leading to changes in the VSD internal salt-bridge network, resulting in a reshaping of the permeation pathway and a significant increase in hydrogen bond connectivity throughout the channel. The total gating charge displacement associated with this transition is consistent with experimental estimates. Molecular docking calculations confirm the proposed mechanism for the inhibitory action of 2-guanidinobenzimidazole (2GBI) derived from electrophysiological measurements and mutagenesis. The depolarized structural model is also consistent with the formation of a metal bridge between residues located in the core of the VSD. Taken together, our results suggest that these structural models are representative of the closed and open states of the Hv1 channel.

Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells.

Expression of the voltage gated proton channel (Hv1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of HV1 expression in 3 out of 8 “triple negative” breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (knock-down; KD) and by eliminating HV1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 Knock-out cells did not show reduced migration or H2O2 release. HV1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in HV1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although HV1 KO and KD affect certain cellular mechanisms differently, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.

The voltage-gated proton channel hHv1 is functionally expressed in human chorion-derived mesenchymal stem cells

The voltage-gated proton channel Hv1 is widely expressed, among others, in immune and cancer cells, it provides an efficient cytosolic H+extrusion mechanism and regulates vital functions such as oxidative burst, migration and proliferation. Here we demonstrate the presence of human Hv1 (hHv1) in the placenta/chorion-derived mesenchymal stem cells (cMSCs) using RT-PCR. The voltage- and pH-dependent gating of the current is similar to that of hHv1 expressed in cell lines and that the current is blocked by 5-chloro-2-guanidinobenzimidazole (ClGBI) and activated by arachidonic acid (AA). Inhibition of hHv1 by ClGBI significantly decreases mineral matrix production of cMSCs induced by conditions mimicking physiological or pathological (inorganic phosphate, Pi) induction of osteogenesis. Wound healing assay and single cell motility analysis show that ClGBI significantly inhibits the migration of cMSCs. Thus, seminal functions of cMSCs are modulated by hHv1 which makes this channel as an attractive target for controlling advantages/disadvantages of MSCs therapy.

Differential expression of the long and truncated Hv1 isoforms in breast-cancer cells.

Metabolic reprogramming of cancer cells results in a high production of acidic substances that must be extruded to maintain tumor-cell viability. The voltage-gated proton channel (Hv1) mediates highly selective effluxes of hydronium-ion (H+ ) that prevent deleterious cytoplasmic acidification. In the work described here, we demonstrated for the first time that the amino-terminal-truncated isoform of Hv1 is more highly expressed in tumorigenic breast-cancer-cell lines than in nontumorigenic breast cells. With respect to Hv1 function, we observed that pharmacologic inhibition of that channel, mediated by the specific blocker 5-chloro-2-guanidinobenzimidazole, produced a drop in intracellular pH and a decrease in cell viability, both in monolayer and in three-dimensional cultures, and adversely affected the cell-cycle in tumorigenic breast cells without altering the cycling of nontumorigenic cells. In conclusion, our results demonstrated that the Hv1 channel could be a potential tool both as a biomarker and as a therapeutic target in breast-cancer disease.

Hv1/VSOP regulates neutrophil directional migration and ERK activity by tuning ROS production.

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils.

Zinc modulation of proton currents in a new voltage-gated proton channel suggests a mechanism of inhibition.

The HV1 voltage‐gated proton (HV1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best‐described physiological inhibitor of HV1 is Zn2+. Externally applied ZnCl2 drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn2+ and Cd2+ on HV1 from Nicoletia phytophila, NpHV1. We introduced mutations at potential Zn2+ coordination sites and measured Zn2+ inhibition in different extracellular pH, with Zn2+ concentrations up to 1000 μm. Zn2+ inhibition in NpHV1 was quantified by the slowing of the activation time constant and a positive shift of the conductance–voltage curve. Replacing aspartate in the S3‐S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage–conductance curve, such that Zn2+ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn2+ in the S3‐S4 linker. A simple Hodgkin Huxley model of NpHV1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high‐resolution clear native gel electrophoresis (hrCNE) confirmed that NpHV1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development.

Role of the time dependence of Boltzmann open probability in voltage-gated proton channels

The modeling and simulation of experimental families of current-time (I-t) curves of dimeric voltage-gated proton channels and of proton-conducting voltage sensing domains (VSDs) with a minimum of free parameters requires the movement of protons to be controlled by the rate of increase of the Boltzmann open probability p over time in passing from the holding to the depolarizing potential. Families of I-t curves of protomers and proton-conducting VSDs can be satisfactorily fitted by the use of a single free parameter expressing the rate constant kp for the increase of p over time. Families of I-t curves of dimeric Hv1 channels can be fitted by a model that assumes an initial proton current I1 flowing along the two monomeric units, while they are still operating separately; I1 is gradually replaced by a slower and more potential-dependent current I2 flowing when the two monomers start operating jointly under the control of the coiled-coil domain. Here too, p is assumed to increase over time with a rate constant kp that doubles in passing from I1 to I2, with fit requiring three free parameters. Chord conductance yields erroneously high gating charges when fitted by the Boltzmann function, differently from slope conductance.

Scorpion toxin inhibits the voltage-gated proton channel using a Zn2+ -like long-range conformational coupling mechanism.

Blocking the voltage-gated proton channel HV 1 is a promising strategy for the treatment of diseases like ischaemia stroke and cancer. However, few HV 1 channel antagonists have been reported. Here, we have identified a novel HV 1 channel antagonist from scorpion venom and have elucidated its action mechanism. HV 1 and NaV channels were heterologously expressed in mammalian cell lines and their currents recorded using whole-cell patch clamp. Site-directed mutagenesis was used to generate mutants. Toxins were recombinantly produced in Escherichia coli. AGAP/W38F-HV 1 interaction was modelled by molecular dynamics simulations. The scorpion toxin AGAP (anti-tumour analgesic peptide) potently inhibited HV 1 currents. One AGAP mutant has reduced NaV channel activity but intact HV 1 activity (AGAP/W38F). AGAP/W38F inhibited HV 1 channel activation by trapping its S4 voltage sensor in a deactivated state and inhibited HV 1 currents with less pH dependence than Zn2+ . Mutation analysis showed that the binding pockets of AGAP/W38F and Zn2+ in HV 1 channel partly overlapped (common sites are His140 and His193). The E153A mutation at the intracellular Coulombic network (ICN) in HV 1 channel markedly reduced AGAP/W38F inhibition, as observed for Zn2+ . Experimental data and MD simulations suggested that AGAP/W38F inhibited HV 1 channel using a Zn2+ -like long-range conformational coupling mechanism. Our results suggest that the Zn2+ binding pocket in HV 1 channel might be a hotspot for modulators and valuable for designing HV 1 channel ligands. Moreover, AGAP/W38F is a useful molecular probe to study HV 1 channel and a lead compound for drug development.

Loss of the voltage-gated proton channel Hv1 decreases insulin secretion and leads to hyperglycemia and glucose intolerance in mice

Insulin secretion by pancreatic islet β-cells is regulated by glucose levels and is accompanied by proton generation. The voltage-gated proton channel Hv1 is present in pancreatic β-cells and extremely selective for protons. However, whether Hv1 is involved in insulin secretion is unclear. Here we demonstrate that Hv1 promotes insulin secretion of pancreatic β-cells and glucose homeostasis. Hv1-deficient mice displayed hyperglycemia and glucose intolerance because of reduced insulin secretion but retained normal peripheral insulin sensitivity. Moreover, Hv1 loss contributed much more to severe glucose intolerance as the mice got older. Islets of Hv1-deficient and heterozygous mice were markedly deficient in glucose- and K+-induced insulin secretion. In perifusion assays, Hv1 deletion dramatically reduced the first and second phase of glucose-stimulated insulin secretion. Islet insulin and proinsulin content was reduced, and histological analysis of pancreas slices revealed an accompanying modest reduction of β-cell mass in Hv1 knockout mice. EM observations also indicated a reduction in insulin granule size, but not granule number or granule docking, in Hv1-deficient mice. Mechanistically, Hv1 loss limited the capacity for glucose-induced membrane depolarization, accompanied by a reduced ability of glucose to raise Ca2+ levels in islets, as evidenced by decreased durations of individual calcium oscillations. Moreover, Hv1 expression was significantly reduced in pancreatic β-cells from streptozotocin-induced diabetic mice, indicating that Hv1 deficiency is associated with β-cell dysfunction and diabetes. We conclude that Hv1 regulates insulin secretion and glucose homeostasis through a mechanism that depends on intracellular Ca2+ levels and membrane depolarization.

Microglial Hv1 exacerbates secondary damage after spinal cord injury in mice

The pathological process of spinal cord injury (SCI) is complex, particularly during secondary damage that triggers a multiphasic glial reaction consisting of both detrimental and beneficial effects. Deletion of a novel voltage-gated proton channel (Hv1) functionally expressed in microglia has been shown to confer neuroprotection during ischemic stroke. Here, we hypothesized that microglial Hv1 may also participate in the process of SCI through modulating glial responses. To test this hypothesis, we employed an SCI model in Hv1-knockout (Hv1−/−) and wild type (WT) mice and assessed resulting microglial polarization, accumulation of pro-inflammatory cytokines, astrocytic activation, oligodendrocytic apoptosis, lesion sizes, and demyelinated areas. Compared with post-SCI results in WT mice, post-SCI Hv1−/− mice exhibited an M2-dominant microglial polarization, decreased accumulation of microglia, and reduced production of pro-inflammatory factors such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β). Additionally, Hv1−/− mice had significantly attenuated reactive astrogliosis and reduced expression of chondroitin sulphate proteoglycans (CSPGs) after SCI. Furthermore, Hv1 deficiency reduced SCI-induced oligodendrocytic apoptosis, demyelinated areas, and cavity formation. Collectively, our results provide the first evidence suggesting that microglial Hv1 may be a multi-mechanism therapeutic target for the treatment of SCI.

Molecular Basis of Voltage-Gated Proton Channel's Intracellular Trafficking

Voltage-gated proton channel (HV1) presents a physiological role of great importance to phagocytosis and sub sequential death of microorganisms in phagocytic cells, such as neutrophils and microglia. Recently various studies describe the contribution of this channel in a higher malignity of tumors and its proliferation. Additionally, HV1 channels have been related to augmented tissue damage in cerebral stroke. Even though these pathological processes have been associated with a higher superficial expression of HV1 channels, little is known about how their abundance in the plasma membrane is regulated. Thus, by combining molecular approaches, in this work we have studied the molecular mechanisms of HV1 channel's intracellular trafficking, associating modifications in its C-terminal domain with its intracellular localization. Therefore, full length human voltage-gated proton channel (hHV1 WT) was fused with the fluorescent protein mCherry and site-directed mutagenesis were made in order to delete the C-terminal domain completely (V220X) or partially (I242X and I256X). Electrophysiological recordings of CHO-K1 cells transfected with hHV1 WT and the C-terminal truncated mutants showed that only the V220X mutant significantly reduced the proton current density. Thus, suggesting that the surface expression of this mutant is compromised. High-resolution microscopy analysis of hHV1 WT intracellular localization through co-transfection with 8 different intracellular organelles markers fused with EGFP resulted in a marked co-localization with Rab11, which is present in recycling endossomes. Furthermore, acute treatment of hHV1 WT transfected CHO-K1 cells with microtubule and actin disruptive agents, nocodazole and cytochalasin D respectively, diminished the proton current density significantly. Overall these results indicate an association of hHV1 WT with recycling endosomes, a key role of both cytoskeletal components and of the C-terminal domain in hHV1 forward trafficking.

Voltage-Gated Proton Channels Exist in the Plasma Membrane of Human Oocytes

Voltage gated proton channels (HV1) are highly expressed in human (but not mouse) sperm where they contribute to capacitation and motility (Lishko et al. 2010. Cell. 140:327; Musset et al. 2012. J. Biol. Chem. 287:9376). We investigated whether HV1 are also present in human oocytes. Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Oocytes were retrieved by transvaginal ovarian follicle aspiration following treatment with recombinant follicle stimulating hormone and purified urinary gonadotropins with GnRH antagonist for controlled ovarian hyperstimulation. After sufficient 18-20 mm follicles were visualized by transvaginal ultrasound, human chorionic gonadotropin was administered to initiate the ovulatory cascade. Transvaginal aspiration of the follicles under ultrasound guidance was performed 36 h later. Human oocytes are large ∼110 μm diameter spheres surrounded by a thick extracellular glycoprotein matrix (the zona pellucida). Their size and zona pellucida make oocytes difficult to study by conventional patch-clamp techniques. To obtain an electrically tight seal we exposed bare oolemma by removing sections of the zona pellucida using a laser, followed by repeated pipetting to further separate the oocyte from the zona pellucida. Patch-clamp studies were performed using whole-cell configuration on oocytes, vesicles derived from oocytes, or inside-out patches of membrane, under conditions optimized for voltage-gated proton currents. Proton currents were present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene. The gating kinetics, voltage dependence, and selectivity were indistinguishable from those of hHV1 in heterologous expression systems. Possible functions for voltage gated proton channels in human oocytes, most likely involving pH regulation, may now be contemplated.

Detecting and Modelling Conformational States of the Proton Channel with Voltage-Clamp Fluorometry

The permeation of protons through the voltage-gated proton channel, Hv1, is regulated by the voltage-dependent movement of a transmembrane (S4) helix containing positively charged arginine residues. To examine the movement of this helix with voltage-clamp fluorometry (VCF) we conjugated an environmentally sensitive fluorophore (TAMRA-MTS) to a cysteine residue (E241C) introduced at the extracellular end of the S4 helix of the Ci-Hv1 proton channel expressed in Xenopus laevis oocytes. In previous work we found that the voltage-dependent fluorescence at this position is largely due to quenching by nearby aromatic amino acids.

The Energy Landscape of Voltage Sensing in Ci-VSP

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane associated phosphatidylinositol lipid phosphatase. The enzymatic activity of the cytoplasmic phosphatase domain is directly regulated by changes in the transmembrane electric field through conformational rearrangements at the voltage-sensing domain (VSD). Ci-VSD shares high sequence and structural similarity with the VSD of voltage-gated cation and proton channels, and is the only membrane-spanning domain of Ci-VSP. Previously, we have determined the structure of Ci-VSD in its resting (Down) and activated (Up) conformations. Thus, this system provides us with a great opportunity to study the energetics of the gating mechanism of this isolated VSD. Using mutagenesis, electrophysiology, modeling and molecular dynamics simulations, we systematically investigated residue-residue interactions between the segments of the VSD and the interactions between the VSD and lipids during a complete functional gating cycle. We found that Ci-VSD can adopt mainly four conformational states: the Up and Down states determined by crystallography, in addition to a further down (‘Down-minus’) and a further up (‘Up-plus’) states. One helix turn translation of the S4 segment drives the transition between two adjacent states. Upon depolarization, the VSD rearranges from the ‘Down-minus’ state to the ‘Up-plus’ state in a sequential stepwise way, translating one positively charged residue across the membrane electric field at each transition, resulting in a gating charge of ∼3 e0. Furthermore, cysteine mutations on the extracellular side of S4 can form metal-ion bridges with lipid phosphate group and decrease the total gating charge displacement with intensity depending on the external Cd2+ concentration, while cysteine mutations on the S1−S3 segments have little effect, representing minor conformational changes of them during gating and do not contribute to the gating charge.