Channel Hv1 Research Articles

Identification of a vacuolar proton channel that triggers the bioluminescent flash in dinoflagellates.

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel (HV1) that opens in response to depolarizing voltage across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thereby causing a pH drop that triggers light emission. HV1 channels were subsequently identified and demonstrated to have important functions in a multitude of eukaryotic cells. Here we report a predicted protein from Lingulodinium polyedrum that displays hallmark properties of bona fide HV1, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. Western blotting and fluorescence confocal microscopy of isolated L. polyedrum scintillons immunostained with antibody to LpHV1 confirm LpHV1’s predicted organellar location. Proteomics analysis demonstrates that isolated scintillon preparations contain peptides that map to LpHV1. Finally, Zn2+ inhibits both LpHV1 proton current and the acid-induced flash in isolated scintillons. These results implicate LpHV1 as the voltage gated proton channel that triggers bioluminescence in L. polyedrum, confirming Hastings’ hypothesis. The same channel likely mediates the action potential that communicates the signal along the tonoplast to the scintillon.

Gating Currents in Hv1 Proton Channels Reveal a Voltage Sensor Mode Shift/Relaxation

The concept of electromechanical coupling between voltage sensor (VS) activation and pore opening is firmly established for tetrameric voltage-gated cation channels (VGCs). Coupling of VS activation to opening of the intrinsic ‘aqueous’ H+ conductance (GAQ) in Hv1 channels is not well understood, in part because of technical difficulties inherent to measuring gating current in isolation from GAQ-mediated ionic current and achieving sufficiently high protein expression. Here we show that a combination of point mutations simultaneously accelerates activation kinetics and blocks outward current carried by GAQ.

Quantum Calculations of Large Segments of a Voltage Sensing Domain of a Voltage Gated Channel

Several large scale calculations of sections of the voltage sensing domain (VSD) of the Kv1.2 channel (pdb: 3Lut), including structure optimizations at HF/6-31G∗ level, were done with applied electric fields of both polarities, or without field. Atoms at the ends of the S1, S2, and S3 segments were frozen. With the S4 segment free to move, the backbone displacement of S4 relative to the S1,S2,S3 block remained below approximately 2 Å in calculations that included the side chains. Thus gating current would not come from the motion of S4 perpendicular to the membrane surface. Optimizations with protons in several possible positions are being carried out, and a possible response of protons to field, in which they move through exchange of partners, is suggested by the energy of the systems in which the protons shift, e.g., from arginine to glutamate. This leaves an unionized salt bridge with energy less than that of the ionized salt bridge (see accompanying Kariev/Green abstract). Proton shifts are required: e.g., calculated open state, salt bridge R300 nitrogen to E226 oxygen distance, with Y266 very close, do not agree with the X-ray structure with the salt bridge ionized. Second, the E183-R297 salt bridge distance corresponds to the X-ray structure, ionized or not, but the neutral case is appreciably lower in energy. Thus this salt bridge is likely to be neutral in the open state. Based on related results, we suggest a hypothesis for a proton pathway that creates the gating current in Kv1.2. It also appears that the Hv1 channel deviates from Kv1.2 near R201/D108/R204(Hv1):R300/E226/R303(Kv1.2), leading to a hypothesis for a path for the protons in Hv1. Water molecules also are an essential part of the path in the upper and lower sections of the VSD.

Oil Slicks Detection From Polarimetric Data Using Stochastic Distances Between Complex Wishart Distributions

Polarimetric synthetic aperture radars (PolSAR) have been used to detect oil slicks at the sea surface. Different techniques to extract information from polarimetric data, using an adequate statistical distribution are currently available. A region-based classifier for PolSAR data - named PolClass - uses a supervised approach to compare stochastic distances between scaled complex Wishart distributions and hypothesis tests to associate confidence levels into the classification results. In this paper, the integrated use of these distances together with the uncertainty maps is applied for the first time to detect oil slicks. A quad-pol Radarsat-2 data, acquired during one open-water controlled exercise, was used to perform this test. The PolClass achieved similar overall accuracies for four stochastic distances, reaching 96.54% of global accuracy, the best result obtained by the Hellinger distance. A comparison between the full- and dual-pol matrices indicated that the results obtained with the VV-HH-HV, HH-HV, and VV-HV polarizations are statistically equivalent, but different from that obtained using the HH-VV. Therefore, the exclusion of the HV channel affected the detection of only mineral oils. The classifier demonstrated the potential to detect the three types of oils released, being more effective in detecting biogenic oils rather than mineral oils. The uncertainty levels increase from the center to the border of the mineral oil slicks, indicating the presence of transition regions, possibly related to different weathering mechanisms. The proposed approach will contribute to the understanding of where different physical and chemical processes may be acting, associating confidence levels to the classification results.

High precision, low disturbance calibration system for the CMS Barrel Electromagnetic Calorimeter High Voltage apparatus

The CMS Electromagnetic Calorimeter utilizes scintillation lead tungstate crystals, with avalanche photodiodes (APD) as photo-detectors in the barrel part. 1224 HV channels bias groups of 50 APD pairs, each at a voltage of about 380 V. The APD gain dependence on the voltage is 3%/V. A stability of better than 60 mV is needed to have negligible impact on the calorimeter energy resolution. Until 2015 manual calibrations were performed yearly. A new calibration system was deployed recently, which satisfies the requirement of low disturbance and high precision. The system is discussed in detail and first operational experience is presented.

The inhibition of voltage-gated H+ channel (HVCN1) induces acidification of leukemic Jurkat T cells promoting cell death by apoptosis.

Cellular energetic deregulation is widely known to produce an overproduction of acidic species in cancer cells. This acid overload must be counterbalanced with a high rate of H+ extrusion to maintain cell viability. In this sense, many H+ transporters have been reported to be crucial for cell survival and proposed as antineoplastic target. By the way, voltage-gated proton channels (Hv1) mediate highly selective H+ outward currents, capable to compensate acid burden in brief periods of time. This structure is canonically described acting as NADPH oxidase counterbalance in reactive oxygen species production. In this work, we show, for the first time in a oncohematologic cell line, that inhibition of Hv1 channels by Zn2+ and the more selective blocker 2-(6-chloro-1H-benzimidazol-2-yl)guanidine (ClGBI) progressively decreases intracellular pH in resting conditions. This acidification is evident minutes after blockade and progresses under prolonged exposure (2, 17, and 48h), and we firstly demonstrate that this is followed by cell death through apoptosis (annexin V binding). Altogether, these results contribute strong evidence that this channel might be a new therapeutic target in cancer.

Coastal Zone Classification With Fully Polarimetric SAR Imagery

Classifying different types of land cover in coastal zones using synthetic aperture radar (SAR) imagery is a challenge due to the fact that many types of coastal zone have similar backscattering characteristics. In this letter, we propose an unsupervised method based on a three-channel joint sparse representation (SR) classification with fully polarimetric SAR (PolSAR) data. The proposed method utilizes both texture and polarimetric feature information extracted from the HH, HV, and VV channels of a SAR image. The texture features are extracted by applying a wavelet transform to a SAR image, and then sparsely represented based on the correlation among the three channels. The polarimetric features, i.e., the scattering entropy and scattering angle from the $H/\alpha$ model, are also sparsely represented. A joint SR algorithm using both texture and polarimetric features is constructed to establish target dictionaries. An orthogonal matching pursuit algorithm is then used to calculate sparse coefficients. Hybrid coefficients are inputted to the kernel support vector machine for a fully PolSAR image classification. We applied the proposed algorithm to an Advanced Land Observing Satellite-2 L-band SAR image acquired in the Yellow River Delta, China. The classified land types are validated against the official survey map. The algorithm performs well in distinguishing six coastal land-use types. A comparison study is also conducted to show that proposed algorithm outperforms two commonly used classification methods.

The Hv1 proton channel responds to mechanical stimuli.

The voltage-gated proton channel, Hv1, is expressed in tissues throughout the body and plays important roles in pH homeostasis and regulation of NADPH oxidase. Hv1 operates in membrane compartments that experience strong mechanical forces under physiological or pathological conditions. In microglia, for example, Hv1 activity is potentiated by cell swelling and causes an increase in brain damage after stroke. The channel complex consists of two proton-permeable voltage-sensing domains (VSDs) linked by a cytoplasmic coiled-coil domain. Here, we report that these VSDs directly respond to mechanical stimuli. We find that membrane stretch facilitates Hv1 channel opening by increasing the rate of activation and shifting the steady-state activation curve to less depolarized potentials. In the presence of a transmembrane pH gradient, membrane stretch alone opens the channel without the need for strong depolarizations. The effect of membrane stretch persists for several minutes after the mechanical stimulus is turned off, suggesting that the channel switches to a "facilitated" mode in which opening occurs more readily and then slowly reverts to the normal mode observed in the absence of membrane stretch. Conductance simulations with a six-state model recapitulate all the features of the channel's response to mechanical stimulation. Hv1 mechanosensitivity thus provides a mechanistic link between channel activation in microglia and brain damage after stroke.

Molecular mechanism of Zn2+ inhibition of a voltage-gated proton channel

Voltage-gated proton (Hv1) channels are involved in many physiological processes, such as pH homeostasis and the innate immune response. Zn2+ is an important physiological inhibitor of Hv1. Sperm cells are quiescent in the male reproductive system due to Zn2+ inhibition of Hv1 channels, but become active once introduced into the low-Zn2+-concentration environment of the female reproductive tract. How Zn2+ inhibits Hv1 is not completely understood. In this study, we use the voltage clamp fluorometry technique to identify the molecular mechanism of Zn2+ inhibition of Hv1. We find that Zn2+ binds to both the activated closed and resting closed states of the Hv1 channel, thereby inhibiting both voltage sensor motion and gate opening. Mutations of some Hv1 residues affect only Zn2+ inhibition of the voltage sensor motion, whereas mutations of other residues also affect Zn2+ inhibition of gate opening. These effects are similar in monomeric and dimeric Hv1 channels, suggesting that the Zn2+-binding sites are localized within each subunit of the dimeric Hv1. We propose that Zn2+ binding has two major effects on Hv1: (i) at low concentrations, Zn2+ binds to one site and prevents the opening conformational change of the pore of Hv1, thereby inhibiting proton conduction; and (ii) at high concentrations, Zn2+, in addition, binds to a second site and inhibits the outward movement of the voltage sensor of Hv1. Elucidating the molecular mechanism of how Zn2+ inhibits Hv1 will further our understanding of Hv1 function and might provide valuable information for future drug development for Hv1 channels.

Voltage-Gated Proton Channel in Human Glioblastoma Multiforme Cells.

Solid tumors tend to have a more glycolytic metabolism leading to an accumulation of acidic metabolites in their cytosol, and consequently, their intracellular pH (pHi) turns critically lower if the cells do not handle the acid excess. Recently, it was proposed that the voltage gated proton channels (HV1) can regulate the pHi in several cancers. Here we report the functional expression of voltage gated proton channels in a human glioblastoma multiforme (GBM) cell line, the most common and lethal brain tumor. T98G cells presented an outward, slow activating voltage-dependent proton current, which was also ΔpH-dependent and inhibited by ZnCl2, characterizing it as being conducted by HV1 channels. Furthermore, blocking HV1 channels with ZnCl2 significantly reduced the pHi, cell survival, and migration, indicating an important role for HV1 for tumor proliferation and progression in GBM. Overall, our results suggest that HV1 channels can be a new therapeutic target for GBM.

Abstract WP274: Inhibition of Na+/H+ Exchanger Isoform 1 and/or Voltage Gated Proton Channel Improves Outcomes in an Embolic Stroke Model

While Na+/H+ exchanger isoform 1 inhibitor (NHEi) has been reported to be neuro- and cardio-protective in experimental stroke and myocardial infarction, respectively, clinical use of NHEi for cardioprotection has stalled due to increased cerebrovascular events. NHEi has been demonstrated to increase Hv1 activity and we recently showed that NHEi activates NADPH oxidase and results in amplified superoxide formation in a voltage gated proton channel Hv1-dependent manner. In the CNS, Hv1 is localized primarily to microglia and deletion of Hv1 is neuroprotective after permanent and transient middle cerebral artery occlusion (MCAO). In the current study, we hypothesized that beneficial effect of NHEi after MCAO will be greater in a rat model lacking Hv1 due to loss of a potentially deleterious increase in Hv1 activation and NADPH oxidase activity. The wild type (WT) and Hv1 knockout (KO) rats (n=4-6) were treated with vehicle or NHEi (KR-32568, 2 mg/kg, i.v.) at 30 min after embolic MCAO. The neurological deficiency, infarct size, HT index, and edema ratio were assessed 3 days after surgery (Table). Compare to WT rats, KO rats had smaller infarct, less edema, and better neurological outcomes as previously found in the suture model. NHEi decreased infarct size and edema in both strains. While there was no significant difference in HT between WT and KO rats, the HT was less in WT rats with NHEi. Functional outcomes were significantly improved with NHEi in WT group, while the KO groups had a trend for a better outcomes with NHEi. These data indicate that NHE inhibition in the acute stroke period is similarly effective in both WT and Hv1 KO animals in providing neurovascular protection. Our data do not support the hypothesis that a deleterious increase in Hv1 dependent NADPH oxidase activity limits the beneficial actions of NHEi in embolic stroke. Further studies are needed to explore the underlying mechanism of the interaction between NHEi and the Hv1 channel in ischemic stroke.

An Insect Proton Channel

Voltage-gated proton channels have been identified in a wide range of eukaryotes. The channel is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. Especially in the animal kingdom voltage gated proton channels can be found consistently. However, most insect genomes analyzed so far seems to be deprived of voltage-gated proton channels. Therefore, it has been proposed that insects do not possess voltage-gated proton channels, although until recently mainly dipteran insect genomes were sequenced. Despite this long standing dogma, here we report the discovery of voltage-gated proton channels in insects. We have performed a comprehensive search in insect TSA data bases and found putative Hv homologs in basal insects and various polyneopteran species. As a prototype insect Hv channel, the proton channel of the zygentoma Nicoletia phytophila (NpHV1) is composed of 239 amino acids with protein identity of 33% to the human proton channel hHV1. Patch clamp measurements in a heterologous expression system showed proton selectivity, pH dependent and voltage dependent gating. Interestingly, NpHv1 shows a slightly enhanced pH dependent gating compared to the human channel. Mutations at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of the aspartate in the first transmembrane segment in all voltage-gated proton channels.

Currents through Hv1 channels deplete protons in their vicinity

Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this work, we directly measure the proton depletion caused by activation of Hv1 proton channels using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Our results provide direct evidence of local proton depletion caused by flux through the proton-selective channel.

Effects of unsaturated fatty acids on the kinetics of voltage-gated proton channels heterologously expressed in cultured cells.

Arachidonic acid (AA) greatly enhances the activity of the voltage-gated proton (Hv) channel, although its mechanism of action and physiological function remain unclear. In the present study, we analysed the effects of AA on proton currents through Hv channels heterologously expressed in HEK293T cells. The dramatic increase in proton current amplitude elicited by AA was accompanied by accelerated activation kinetics and a leftward shift in the voltage-dependence of activation. Mutagenesis studies suggest the two aforementioned effects of AA reflect two distinct structural mechanisms. Application of phospholipase A2 , which liberates AA from phospholipids in the membrane, also enhances Hv channel activity, supporting the idea that AA modulates Hv channel activity within physiological contexts. Unsaturated fatty acids are key components of the biological membranes of all cells, and precursors of mediators for cell signalling. Arachidonic acid (AA) is an unsaturated fatty acid known to modulate the activities of various ion channels, including the voltage-gated proton (Hv) channel, which supports the rapid production of reactive oxygen species (ROS) in phagocytes through regulation of pH and membrane potential. However, the molecular mechanisms and physiological functions of the effects of AA on Hv channels remain unclear. In the present study, we report an electrophysiological analysis of the effects of AA on the mouse Hv channel (mHv1) heterologously expressed in HEK293T cells. Application of AA to excised inside-out patch membranes rapidly induced a robust increase in the amplitude of the proton current through mHv1. The current increase was accompanied by accelerated activation kinetics and a small leftward shift of the current-voltage relationship. In monomeric channels lacking the coiled-coil region of the channel protein, the shift in the current-voltage relationship was diminished but activation and deactivation remained accelerated. Studies with several AA derivatives showed that double bonds and hydrophilic head groups are essential for the effect of AA, although charge was not important. The application of phospholipase A2 (PLA2), which generates AA from cell membrane phospholipids, stimulated mHv1 activity to a similar extent as direct application of ∼ 20 μM AA, suggesting that endogenous AA may regulate Hv channel activity.

Estimation of Tropical Forest Structural Characteristics Using ALOS-2 SAR Data

The potential of ALOS-2 SAR data for the estimation of tropical forest structural characteristics was assessed in Vietnam by collecting forest inventory data. The effect of polarization and seasonality of the SAR data on the estimation of forest biomass was analyzed. The combination of HH, HV, and HH/HV polarizations using multiple linear regression did not improve the estimation of biomass compared to using the HV channel independently, as the HH and HH/HV variables were not statistically significant. The dry season HV backscattering intensity was highly sensitive to the biomass compared to the rainy season backscattering intensity. The SAR data acquired in the rainy season with humid and wet canopies was not very sensitive to the biomass. The strong dependence of the biomass estimates with the season of SAR data acquisition confirmed that the choice of right season SAR data is very important for improving the satellite based estimates of the biomass. The validation results showed that the dry season HV polarization could explain 54% variation of the biomass.

Interrogation of the intersubunit interface of the open Hv1 proton channel with a probe of allosteric coupling

The Hv1 voltage-gated proton channel is a dimeric complex consisting of two voltage-sensing domains (VSDs), each containing a gated proton permeation pathway. Dimerization is controlled by a cytoplasmic coiled-coil domain. The transitions from the closed to the open state in the two VSDs are known to occur cooperatively; however, the underlying mechanism is poorly understood. Intersubunit interfaces play a critical role in allosteric processes; but, such interfaces have not been determined in the open Hv1 channel. Here we show that 2-guanidinothiazole derivatives block the two Hv1 VSDs in a cooperative way, and use one of the compounds as a probe of allosteric coupling between open subunits. We find that the extracellular ends of the first transmembrane segments of the VSDs form the intersubunit interface that mediates coupling between binding sites, while the coiled-coil domain does not directly participate in the process. We also find strong evidence that the channel’s proton selectivity filter controls blocker binding cooperativity.

The Voltage-Gated Proton Channel: A Riddle, Wrapped in a Mystery, inside an Enigma.

The main properties of the voltage-gated proton channel (HV1) are described in this review, along with what is known about how the channel protein structure accomplishes its functions. Just as protons are unique among ions, proton channels are unique among ion channels. Their four transmembrane helices sense voltage and the pH gradient and conduct protons exclusively. Selectivity is achieved by the unique ability of H3O(+) to protonate an Asp-Arg salt bridge. Pathognomonic sensitivity of gating to the pH gradient ensures HV1 channel opening only when acid extrusion will result, which is crucial to most of its biological functions. An exception occurs in dinoflagellates in which influx of H(+) through HV1 triggers the bioluminescent flash. Pharmacological interventions that promise to ameliorate cancer, asthma, brain damage in ischemic stroke, Alzheimer's disease, autoimmune diseases, and numerous other conditions await future progress.

The voltage-gated proton channel Hv1/VSOP inhibits neutrophil granule release.

Neutrophil granule exocytosis is crucial for host defense and inflammation. Neutrophils contain 4 types of granules, the exocytotic release of which is differentially regulated. This exocytosis is known to be driven by diverse mediators, including calcium and nucleotides, but the precise molecular mechanism remains largely unknown. We show in the present study that voltage-gated proton (Hv) channels are necessary for the proper release of azurophilic granules in neutrophils. On activation of NADPH oxidase by PMA and IgG, neutrophils derived from Hvcn1 gene knockout mouse exhibited greater secretion of MPO and elastase than WT cells. In contrast, release of LTF enriched in specific granules was not enhanced in these cells. The excess release of azurophilic granules in Hv1/VSOP-deficient neutrophils was suppressed by inhibiting NADPH oxidase activity and, in part, by valinomycin, a potassium ionophore. In addition, Hv1/VSOP-deficient mice exhibited more severe lung inflammation after intranasal Candida albicans infection than WT mice. These findings suggest that the Hv channel acts to specifically dampen the release of azurophilic granules through, in part, the suppression of increased positive charges at the plasma membrane accompanied by the activation of NADPH oxidase in neutrophils.

Selectivity Mechanism of the Voltage-gated Proton Channel, HV1.

Voltage-gated proton channels, HV1, trigger bioluminescence in dinoflagellates, enable calcification in coccolithophores, and play multifarious roles in human health. Because the proton concentration is minuscule, exquisite selectivity for protons over other ions is critical to HV1 function. The selectivity of the open HV1 channel requires an aspartate near an arginine in the selectivity filter (SF), a narrow region that dictates proton selectivity, but the mechanism of proton selectivity is unknown. Here we use a reduced quantum model to elucidate how the Asp–Arg SF selects protons but excludes other ions. Attached to a ring scaffold, the Asp and Arg side chains formed bidentate hydrogen bonds that occlude the pore. Introducing H3O+ protonated the SF, breaking the Asp–Arg linkage and opening the conduction pathway, whereas Na+ or Cl– was trapped by the SF residue of opposite charge, leaving the linkage intact, thus preventing permeation. An Asp–Lys SF behaved like the Asp–Arg one and was experimentally verified to be proton-selective, as predicted. Hence, interacting acidic and basic residues form favorable AspH0–H2O0–Arg+ interactions with hydronium but unfavorable Asp––X–/X+–Arg+ interactions with anions/cations. This proposed mechanism may apply to other proton-selective molecules engaged in bioenergetics, homeostasis, and signaling.

Abstract T P229: Deletion of Voltage Gated Proton Channel in Microglial Cells is Neurovascular Protective After Ischemic Brain Injury

Despite the failure of antioxidant treatments in clinical trials, the undoubted role of reactive oxygen species (ROS) in neurovascular damage after ischemic stroke calls for a more targeted approach. ROS production by microglia, the primary resident immune cells in the brain, is a key event of this process in ischemic stroke. Voltage gated proton channel, Hv1, is localized primarily to microglia and sustains NADPH oxidase activity. Deletion of Hv1 is neuroprotective after permanent middle cerebral artery occlusion (MCAO). We hypothesized that Hv1-mediated microglial ROS generation is also critical for vascular integrity and contributes to reperfusion injury after transient ischemic stroke. The wildtype (WT) and Hv1 knockout (KO) rats (n=4) were subjected to permanent or 3/24 h transient MCAO. The neurological deficiency, infarct, hemorrhagic transformation, and edema ratio were assessed. We found that in both permanent and transient MCAO model, KO rats develop smaller infarct, less vascular injury, edema, and hemorrhagic transformation, resulting in better short-term functional outcome. These results suggest that deletion of microglial Hv1 channel is vasculoprotective after ischemia/reperfusion and the underlying mechanisms need to be further studied.