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Thomas E Decoursey - One of the best experts on this subject based on the ideXlab platform.
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Expression and function of voltage gated Proton Channels (Hv1) in MDA-MB-231 cells.
PloS one, 2020Co-Authors: Dan J. Bare, Thomas E Decoursey, Vladimir V Cherny, Abde M. Abukhdeir, Deri MorganAbstract: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.
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Voltage-Gated Proton Channels exist in the plasma membrane of human oocytes
Human reproduction (Oxford England), 2019Co-Authors: R Ya Smith, Deri Morgan, Vladimir V Cherny, L Sharma, N Tidswell, M W Molo, Thomas E DecourseyAbstract:Study question Do human oocytes express Voltage-Gated Proton Channels? Summary answer Human oocytes exhibit Voltage-Gated Proton currents. What is known already Voltage-Gated Proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. Study design, size, duration Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. Participants/materials, setting, methods Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect Voltage-Gated Proton currents. Main results and the role of chance Proton currents are 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 that codes for the Voltage-Gated Proton Channel. Large scale data N/A. Limitations, reasons for caution Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. Wider implications of the findings Possible functions for Voltage-Gated Proton Channels in human oocytes may now be contemplated. Study funding/competing interest(s) NIH R35GM126902 (TED), Bears Care (DM). No competing interests. Trial registration number N/A.
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Hydrophobic gasket mutation produces gating pore currents in closed human Voltage-Gated Proton Channels.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Richard L. Banh, Susan M.e. Smith, Deri Morgan, Boris Musset, Vladimir V Cherny, Kethika Kulleperuma, Régis Pomès, Sarah Thomas, Thomas E DecourseyAbstract:The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most Voltage-Gated ion Channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the Channel opens. S4 movement may occur during gating of the human Voltage-Gated Proton Channel, hHV1, but Proton current flows through the same pore in open Channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair Proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated Channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the Channel leaked Protons. Closed-Channel Proton current was smaller than open-Channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed Channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several Channelopathies.
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Gating currents indicate complex gating of Voltage-Gated Proton Channels.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Thomas E DecourseyAbstract:The Voltage-Gated Proton Channel (HV1) is a unique molecule that resides at the interface between ion Channels and bioenergetic molecules that use Proton gradients to store or transduce energy. HV1 plays key roles in the health and disease of diverse tissues and species (1). Important information regarding the physical components of ion-Channel gating (opening and closing, which in turn activate or terminate flow of ionic current through the pore) can be obtained by measuring the size, kinetics, and voltage dependence of gating currents. Fig. 1 illustrates different mechanisms by which gating currents might be generated. In their landmark voltage-clamp studies of sodium and potassium Channels in squid axons in the mid-20th century, Hodgkin and Huxley (2) deduced that ( i ) ionic current flowed through discreet sites (Channels) in the membrane; ( ii ) these permeation pathways were gated, opening and closing in response to changes in membrane potential; and ( iii ) this process most likely involved the movement of charged groups across part or all of the transmembrane electric field. They recognized that the movement of charges within the electric field should generate a small capacitive current, but it was smaller than they could detect. The predicted gating currents were measured in sodium Channels two decades later (3⇓⇓–6). Sodium-Channel gating currents were ∼300 times smaller than the ionic current and were mostly over in 1 ms (3). Several properties of Proton Channels frustrated attempts to measure their gating currents until recently. Carmona et al. (7) report gating currents in HV1 from the sea squirt Ciona intestinalis . A contemporaneous study by De La Rosa and Ramsey (8) reports gating currents in human HV1. The clever experimental approaches devised by these groups to overcome the intransigence of HV1 and their conclusions … [↵][1]1Email: tdecours{at}rush.edu. [1]: #xref-corresp-1-1
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Histidine168 is crucial for ΔpH-dependent gating of the human Voltage-Gated Proton Channel, hHV1.
The Journal of general physiology, 2018Co-Authors: Vladimir V Cherny, Susan M.e. Smith, Deri Morgan, Sarah Thomas, Thomas E DecourseyAbstract:We recently identified a Voltage-Gated Proton Channel gene in the snail Helisoma trivolvis, HtHV1, and determined its electrophysiological properties. Consistent with early studies of Proton currents in snail neurons, HtHV1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pHi). The H+ conductance (gH)-V relationship in the Voltage-Gated Proton Channel (HV1) from other species shifts 40 mV when either pHi or pHo (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of HV1 in many species and in numerous human tissues. The HtHV1 Channel exhibits normal pHo dependence but anomalously weak pHi dependence. In this study, we show that a single point mutation in human hHV1-changing His168 to Gln168, the corresponding residue in HtHV1-compromises the pHi dependence of gating in the human Channel so that it recapitulates the HtHV1 response. This location was previously identified as a contributor to the rapid gating kinetics of HV1 in Strongylocentrotus purpuratus His168 mutation in human HV1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pHo dependence, but changing pHi shifts the gH-V relationship on average by
Yasushi Okamura - One of the best experts on this subject based on the ideXlab platform.
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Supplemental Movies: Real-time functional analysis of Hv1 Channel in neutrophils: a new approach from zebrafish model
2019Co-Authors: Adisorn Ratanayotha, Takafumi Kawai, Yasushi OkamuraAbstract:Voltage-Gated Proton Channel (Hv1) has been studied in various immune cells, including neutrophils. However, most studies have taken in vitro approach using isolated cells or primary cultured cells of mammals; therefore, limited evidence is available on the function of Hv1 in a physiological context. In this study, we have developed the in vivo system that enables real-time functional analysis of Hv1 using zebrafish embryos (Danio rerio). Hvcn1-deficiency (hvcn1-/-) in zebrafish completely abolished Voltage-Gated Proton current which is typically observed in wild type neutrophils. Importantly, hvcn1-deficiency significantly reduced ROS production and calcium response of zebrafish neutrophils, comparable to the results observed in mammalian models. These findings verify zebrafish Hv1 (DrHv1) as the primary contributor for native Hv1-derived Proton current in neutrophils and suggest the conserved function of Hv1 in the immune cells across vertebrate animals. Taking advantage of Hv1 zebrafish model, we compared real-time behaviors of neutrophils between wild type and hvcn1-/- zebrafish in response to tissue injury and acute bacterial infection. Notably, we observed a significant increase in the number of phagosomes in hvcn1-/- neutrophils, raising a possible link between Hv1 and phagosomal maturation. Furthermore, survival analysis of zebrafish larvae potentially supports a protective role of Hv1 in the innate immune response against systemic bacterial infection. This study represents the influence of Hv1 on neutrophil behaviors and highlights the benefits of in vivo approach towards the understanding of Hv1 in a physiological context.Supplemental Movie S1: Representative tracks of WT neutrophil migrations in response to ventral fin injury. Neutrophils, green. Supplemental Movie S2: Representative tracks of hvcn1-/- neutrophil migrations in response to ventral fin injury. Neutrophils, green. Supplemental Movie S3: Representative real-time imaging of WT neutrophil migrations in response to local bacterial infection. Neutrophils, green; bacteria, red.Supplemental Movie S4: Representative tracks of WT neutrophil migrations in response to local bacterial infection. Neutrophils, green. Each colored line represents the course of migration for each neutrophil. The movie is identical to Movie S3, except the images of DsRed+ E. coli were subtracted from these samples to enhance visibility of the tracking courses of neutrophils. Supplemental Movie S5: Representative real-time imaging of hvcn1-/- neutrophil migrations in response to local bacterial infection. Neutrophils, green; bacteria, red. Supplemental Movie S6: Representative tracks of hvcn1-/- neutrophil migrations in response to local bacterial infection. Neutrophils, green. Each colored line represents the course of migration for each neutrophil. The movie is identical to Movie S5, except the images of DsRed+ E. coli were subtracted from these samples to enhance visibility of the tracking courses of neutrophils.
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Zn2+-Binding to the Voltage-Gated Proton Channel Hv1/VSOP
The journal of physical chemistry. B, 2018Co-Authors: Masayo Iwaki, Yasushi Okamura, Kohei Takeshita, Hiroko Kondo, Kengo Kinoshita, Yu Takano, Atsushi Nakagawa, Hideki KandoriAbstract:The Voltage-Gated Proton Channel (Hv1/VSOP) is inhibited by Zn2+, of which the binding site is located in the extracellular region. We utilized attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopy to examine the coordination structure by monitoring protein structural changes induced by Zn2+-binding. The Zn2+-induced difference ATR-FTIR spectra of Hv1 showed IR features that can be assigned to the histidine C5–N1 and carboxylate-COO– stretches as well as amide I changes likely in α-helical peptide bonds. Analysis of vibrational frequencies indicated that the Zn2+ is coordinated by the anionic carboxylate with monodentate mode and by the histidine at N1 (Nτ) position of the neutral imidazole form. Combined with quantum chemical calculations, the most probable coordination structure was proposed as a tetrahedral geometry with ligands of carboxylate and imidazole groups in addition to a water molecule.
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Zn2+-Binding to the Voltage-Gated Proton Channel Hv1/VSOP
2018Co-Authors: Masayo Iwaki, Yasushi Okamura, Kohei Takeshita, Kengo Kinoshita, Yu Takano, Atsushi Nakagawa, Hiroko X. Kondo, Hideki KandoriAbstract:The Voltage-Gated Proton Channel (Hv1/VSOP) is inhibited by Zn2+, of which the binding site is located in the extracellular region. We utilized attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopy to examine the coordination structure by monitoring protein structural changes induced by Zn2+-binding. The Zn2+-induced difference ATR-FTIR spectra of Hv1 showed IR features that can be assigned to the histidine C5–N1 and carboxylate-COO– stretches as well as amide I changes likely in α-helical peptide bonds. Analysis of vibrational frequencies indicated that the Zn2+ is coordinated by the anionic carboxylate with monodentate mode and by the histidine at N1 (Nτ) position of the neutral imidazole form. Combined with quantum chemical calculations, the most probable coordination structure was proposed as a tetrahedral geometry with ligands of carboxylate and imidazole groups in addition to a water molecule
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Molecular and functional characterization of the voltage‐gated Proton Channel in zebrafish neutrophils
Physiological reports, 2017Co-Authors: Adisorn Ratanayotha, Takafumi Kawai, Shin-ichi Higashijima, Yasushi OkamuraAbstract:Voltage-Gated Proton Channels (Hv1/VSOP) are expressed in various cells types, including phagocytes, and are involved in diverse physiological processes. Although hvcn1, the gene encoding Hv1, has been identified across a wide range of species, most of the knowledge about its physiological function and expression profile is limited to mammals. In this study, we investigated the basic properties of DrHv1, the Hv1 ortholog in zebrafish (Danio rerio) which is an excellent animal model owing to the transparency, as well as its functional expression in native cells. Electrophysiological analysis using a heterologous expression system confirmed the properties of a Voltage-Gated Proton Channel are conserved in DrHv1 with differences in threshold and activation kinetics as compared to mouse (Mus musculus) Hv1 (mHv1). RT-PCR analysis revealed that hvcn1 is expressed in zebrafish neutrophils, as is the case in mammals. Subsequent electrophysiological analysis confirmed the functional expression of DrHv1 in zebrafish neutrophils, which suggests Hv1 function in phagocytes is conserved among vertebrates. We also found that DrHv1 is comparatively resistant to extracellular Zn2+, which is a potent inhibitor of mammalian Hv1, and this phenomenon appears to reflect variation in the Zn2+-coordinating residue (histidine) within the extracellular linker region in mammalian Hv1. Notably, the serum Zn2+ concentration is much higher in zebrafish than in mouse, raising the possibility that Zn2+ sensitivity was acquired in accordance with a change in the serum Zn2+ concentration. This study highlights the biological variation and importance of Hv1 in different animal species.
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Comparison between mouse and sea urchin orthologs of Voltage-Gated Proton Channel suggests role of S3 segment in activation gating.
Biochimica et biophysica acta, 2016Co-Authors: Souhei Sakata, Yuichiro Fujiwara, Akira Kawanabe, Tatsuki Kurokawa, Nana Miyawaki, Thomas J. Mccormack, Hiroki Arima, Nurdan Özkucur, Yuka Jinno, Yasushi OkamuraAbstract:The Voltage-Gated Proton Channel, Hv1, is expressed in blood cells, airway epithelium, sperm and microglia, playing important roles in diverse biological contexts including phagocytosis or sperm maturation through its regulation of membrane potential and pH. The gene encoding Hv1, HVCN1, is widely found across many species and is also conserved in unicellular organisms such as algae or dinoflagellates where Hv1 plays role in calcification or bioluminescence. Voltage-Gated Proton Channels exhibit a large variation of activation rate among different species. Here we identify an Hv1 ortholog from sea urchin, Strongylocentrotus purpuratus, SpHv1. SpHv1 retains most of key properties of Hv1 but exhibits 20-60 times more rapid activation kinetics than mammalian orthologs upon heterologous expression in HEK293T cells. Comparison between SpHv1 and mHv1 highlights novel roles of the third transmembrane segment S3 in activation gating of Hv1.
Deri Morgan - One of the best experts on this subject based on the ideXlab platform.
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Expression and function of voltage gated Proton Channels (Hv1) in MDA-MB-231 cells.
PloS one, 2020Co-Authors: Dan J. Bare, Thomas E Decoursey, Vladimir V Cherny, Abde M. Abukhdeir, Deri MorganAbstract: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.
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Voltage-Gated Proton Channels exist in the plasma membrane of human oocytes
Human reproduction (Oxford England), 2019Co-Authors: R Ya Smith, Deri Morgan, Vladimir V Cherny, L Sharma, N Tidswell, M W Molo, Thomas E DecourseyAbstract:Study question Do human oocytes express Voltage-Gated Proton Channels? Summary answer Human oocytes exhibit Voltage-Gated Proton currents. What is known already Voltage-Gated Proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. Study design, size, duration Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. Participants/materials, setting, methods Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect Voltage-Gated Proton currents. Main results and the role of chance Proton currents are 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 that codes for the Voltage-Gated Proton Channel. Large scale data N/A. Limitations, reasons for caution Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. Wider implications of the findings Possible functions for Voltage-Gated Proton Channels in human oocytes may now be contemplated. Study funding/competing interest(s) NIH R35GM126902 (TED), Bears Care (DM). No competing interests. Trial registration number N/A.
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Hydrophobic gasket mutation produces gating pore currents in closed human Voltage-Gated Proton Channels.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Richard L. Banh, Susan M.e. Smith, Deri Morgan, Boris Musset, Vladimir V Cherny, Kethika Kulleperuma, Régis Pomès, Sarah Thomas, Thomas E DecourseyAbstract:The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most Voltage-Gated ion Channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the Channel opens. S4 movement may occur during gating of the human Voltage-Gated Proton Channel, hHV1, but Proton current flows through the same pore in open Channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair Proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated Channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the Channel leaked Protons. Closed-Channel Proton current was smaller than open-Channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed Channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several Channelopathies.
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Histidine168 is crucial for ΔpH-dependent gating of the human Voltage-Gated Proton Channel, hHV1.
The Journal of general physiology, 2018Co-Authors: Vladimir V Cherny, Susan M.e. Smith, Deri Morgan, Sarah Thomas, Thomas E DecourseyAbstract:We recently identified a Voltage-Gated Proton Channel gene in the snail Helisoma trivolvis, HtHV1, and determined its electrophysiological properties. Consistent with early studies of Proton currents in snail neurons, HtHV1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pHi). The H+ conductance (gH)-V relationship in the Voltage-Gated Proton Channel (HV1) from other species shifts 40 mV when either pHi or pHo (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of HV1 in many species and in numerous human tissues. The HtHV1 Channel exhibits normal pHo dependence but anomalously weak pHi dependence. In this study, we show that a single point mutation in human hHV1-changing His168 to Gln168, the corresponding residue in HtHV1-compromises the pHi dependence of gating in the human Channel so that it recapitulates the HtHV1 response. This location was previously identified as a contributor to the rapid gating kinetics of HV1 in Strongylocentrotus purpuratus His168 mutation in human HV1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pHo dependence, but changing pHi shifts the gH-V relationship on average by
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Exotic properties of a Voltage-Gated Proton Channel from the snail Helisoma trivolvis.
The Journal of general physiology, 2018Co-Authors: Sarah Thomas, Susan M.e. Smith, Deri Morgan, Vladimir V Cherny, Liana Artinian, Vincent Rehder, Thomas E DecourseyAbstract:Voltage-Gated Proton Channels, HV1, were first reported in Helix aspersa snail neurons. These H+ Channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the Proton conductance-voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 Channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 Channel conducting inward currents and perhaps mediating a Proton action potential. The anomalous ΔpH-dependent gating of HtHV1 Channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).
Vladimir V Cherny - One of the best experts on this subject based on the ideXlab platform.
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Expression and function of voltage gated Proton Channels (Hv1) in MDA-MB-231 cells.
PloS one, 2020Co-Authors: Dan J. Bare, Thomas E Decoursey, Vladimir V Cherny, Abde M. Abukhdeir, Deri MorganAbstract: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.
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Voltage-Gated Proton Channels exist in the plasma membrane of human oocytes
Human reproduction (Oxford England), 2019Co-Authors: R Ya Smith, Deri Morgan, Vladimir V Cherny, L Sharma, N Tidswell, M W Molo, Thomas E DecourseyAbstract:Study question Do human oocytes express Voltage-Gated Proton Channels? Summary answer Human oocytes exhibit Voltage-Gated Proton currents. What is known already Voltage-Gated Proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. Study design, size, duration Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. Participants/materials, setting, methods Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect Voltage-Gated Proton currents. Main results and the role of chance Proton currents are 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 that codes for the Voltage-Gated Proton Channel. Large scale data N/A. Limitations, reasons for caution Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. Wider implications of the findings Possible functions for Voltage-Gated Proton Channels in human oocytes may now be contemplated. Study funding/competing interest(s) NIH R35GM126902 (TED), Bears Care (DM). No competing interests. Trial registration number N/A.
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Hydrophobic gasket mutation produces gating pore currents in closed human Voltage-Gated Proton Channels.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Richard L. Banh, Susan M.e. Smith, Deri Morgan, Boris Musset, Vladimir V Cherny, Kethika Kulleperuma, Régis Pomès, Sarah Thomas, Thomas E DecourseyAbstract:The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most Voltage-Gated ion Channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the Channel opens. S4 movement may occur during gating of the human Voltage-Gated Proton Channel, hHV1, but Proton current flows through the same pore in open Channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair Proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated Channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the Channel leaked Protons. Closed-Channel Proton current was smaller than open-Channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed Channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several Channelopathies.
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Histidine168 is crucial for ΔpH-dependent gating of the human Voltage-Gated Proton Channel, hHV1.
The Journal of general physiology, 2018Co-Authors: Vladimir V Cherny, Susan M.e. Smith, Deri Morgan, Sarah Thomas, Thomas E DecourseyAbstract:We recently identified a Voltage-Gated Proton Channel gene in the snail Helisoma trivolvis, HtHV1, and determined its electrophysiological properties. Consistent with early studies of Proton currents in snail neurons, HtHV1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pHi). The H+ conductance (gH)-V relationship in the Voltage-Gated Proton Channel (HV1) from other species shifts 40 mV when either pHi or pHo (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of HV1 in many species and in numerous human tissues. The HtHV1 Channel exhibits normal pHo dependence but anomalously weak pHi dependence. In this study, we show that a single point mutation in human hHV1-changing His168 to Gln168, the corresponding residue in HtHV1-compromises the pHi dependence of gating in the human Channel so that it recapitulates the HtHV1 response. This location was previously identified as a contributor to the rapid gating kinetics of HV1 in Strongylocentrotus purpuratus His168 mutation in human HV1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pHo dependence, but changing pHi shifts the gH-V relationship on average by
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Exotic properties of a Voltage-Gated Proton Channel from the snail Helisoma trivolvis.
The Journal of general physiology, 2018Co-Authors: Sarah Thomas, Susan M.e. Smith, Deri Morgan, Vladimir V Cherny, Liana Artinian, Vincent Rehder, Thomas E DecourseyAbstract:Voltage-Gated Proton Channels, HV1, were first reported in Helix aspersa snail neurons. These H+ Channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the Proton conductance-voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 Channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 Channel conducting inward currents and perhaps mediating a Proton action potential. The anomalous ΔpH-dependent gating of HtHV1 Channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).
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Hydrophobic gasket mutation produces gating pore currents in closed human Voltage-Gated Proton Channels.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Richard L. Banh, Susan M.e. Smith, Deri Morgan, Boris Musset, Vladimir V Cherny, Kethika Kulleperuma, Régis Pomès, Sarah Thomas, Thomas E DecourseyAbstract:The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most Voltage-Gated ion Channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the Channel opens. S4 movement may occur during gating of the human Voltage-Gated Proton Channel, hHV1, but Proton current flows through the same pore in open Channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair Proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated Channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the Channel leaked Protons. Closed-Channel Proton current was smaller than open-Channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed Channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several Channelopathies.
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Identification of an HV1 voltage‐gated Proton Channel in insects
The FEBS journal, 2016Co-Authors: Gustavo Chaves, Christian Derst, Arne Franzen, Yuta Mashimo, Ryuichiro Machida, Boris MussetAbstract:UNLABELLED The Voltage-Gated Proton Channel 1 (HV 1) is an important component of the cellular Proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV 1 Channel. In the present study, we report the existence of an HV 1-type Proton Channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV 1 orthologues in various polyneopteran insects. To confirm that these putative HV 1 orthologues were functional Channels, we studied the HV 1 Channel of Nicoletia phytophila (NpHV 1), an insect of the Zygentoma order, in more detail. NpHV 1 comprises 239 amino acids and is 33% identical to the human Voltage-Gated Proton Channel 1. Patch clamp measurements in a heterologous expression system showed Proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, NpHV 1 shows slightly enhanced pH-dependent gating compared to the human Channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of Proton-selective conduction, confirming the importance of this aspartate residue in Voltage-Gated Proton Channels. DATABASE Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722.
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Tryptophan 207 is crucial to the unique properties of the human Voltage-Gated Proton Channel, hHV1.
The Journal of general physiology, 2015Co-Authors: Vladimir V Cherny, Susan M.e. Smith, Deri Morgan, Boris Musset, Gustavo Chaves, Thomas E DecourseyAbstract:Part of the "signature sequence" that defines the Voltage-Gated Proton Channel (H(V1)) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H(V1) genes. Replacing Trp207 in human HV1 (hH(V1)) with Ala, Ser, or Phe facilitated gating, accelerating Channel opening by 100-fold, and closing by 30-fold. Mutant Channels opened at more negative voltages than wild-type (WT) Channels, indicating that in WT Channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for Channel opening decreased to 22 kcal/mol from 30-38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hH(V1). Cation-π interaction between Trp207 and Arg211 evidently latches the Channel closed. Trp207 mutants lost Proton selectivity at pHo >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H(V1) that is essential to its biological functions, was compromised. In the WT hH(V1), ΔpH-dependent gating is shown to saturate above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp207 mutants, ΔpH-dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H(V1) from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pHo and pHi in H(V1) of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow Channel opening, highly temperature-dependent gating kinetics, Proton selectivity, and ΔpH-dependent gating.
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Selectivity Mechanism of the Voltage-Gated Proton Channel, HV1.
Scientific reports, 2015Co-Authors: Todor Dudev, Susan M.e. Smith, Deri Morgan, Boris Musset, Vladimir V Cherny, Thomas E Decoursey, Karine Mazmanian, Carmay LimAbstract: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.
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Enhanced activation of an amino-terminally truncated isoform of the Voltage-Gated Proton Channel HVCN1 enriched in malignant B cells.
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Elayne Hondares, Deri Morgan, Boris Musset, Vladimir V Cherny, Mark A. Brown, Christina Taubert, Mandeep K Bhamrah, David Coe, Federica M. Marelli-berg, John G. GribbenAbstract:HVCN1 (Hydrogen Voltage-Gated Channel 1) is the only mammalian Voltage-Gated Proton Channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr29. Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.
