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1.
FEBS J ; 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32160407

RESUMO

The HV 1 voltage-gated proton (HV 1) 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 HV 1 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 trivolis and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn2+ and Cd2+ on HV 1 from Nicoletia phytophila, NpHV 1. 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 NpHV 1 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 NpHV 1 suggests a decrease of the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high resolution clear native gel electrophoresis (hrCNE) confirmed that NpHV 1 functions as a dimer. The data supports the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development.

2.
Biophys J ; 118(5): 1221-1233, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-31972155

RESUMO

Voltage-gated proton channels (HV1) are essential for various physiological tasks but are strongly inhibited by Zn2+ cations. Some determinants of Zn2+ binding have been elucidated experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn2+ binds to monomeric HV1 despite evidence that HV1 expresses as a dimer and that the dimer has a higher affinity for zinc than the monomer and experimental data that suggest coordination in the dimer interface. The results of former studies are also controversial, e.g., supporting either one single or two binding sites. Some structural determinants of the binding are still elusive. We performed a series of molecular dynamics simulations to address different structures of the human proton channel, the monomer and two plausible dimer conformations, to compare their respective potential to interact with and bind Zn2+ via the essential histidines. The series consisted of several copies of the system to generate independent trajectories and increase the significance compared to a single simulation. The amount of time simulated totals 29.9 µs for 126 simulations of systems comprising ∼59,000 to ∼187,000 atoms. Our approach confirms the existence of two binding sites in monomeric and dimeric human HV1. The dimer interface is more efficient for attracting and binding Zn2+ via the essential histidines than the monomer or a dimer with the histidines in the periphery. The higher affinity is due to the residues in the dimer interface that create an attractive electrostatic potential funneling the zinc cations toward the binding sites.

3.
Proc Natl Acad Sci U S A ; 116(38): 18951-18961, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31462498

RESUMO

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.

4.
Pflugers Arch ; 470(7): 1017-1033, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29717355

RESUMO

Neutrophil granulocytes are exposed to widely varying microenvironmental conditions when pursuing their physiological or pathophysiological functions such as fighting invading bacteria or infiltrating cancer tissue. Examples for harsh environmental challenges include among others mechanical shear stress during the recruitment from the vasculature or the hypoxic and acidotic conditions within the tumor microenvironment. Chemokine gradients, reactive oxygen species, pressure, matrix elasticity, and temperature can be added to the list of potential challenges. Transient receptor potential (TRP) channels serve as cellular sensors since they respond to many of the abovementioned environmental stimuli. The present review investigates the role of TRP channels in neutrophil granulocytes and their role in regulating and adapting neutrophil function to microenvironmental cues. Following a brief description of neutrophil functions, we provide an overview of the electrophysiological characterization of neutrophilic ion channels. We then summarize the function of individual TRP channels in neutrophil granulocytes with a focus on TRPC6 and TRPM2 channels. We close the review by discussing the impact of the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) on neutrophil granulocytes. Since neutrophil infiltration into PDAC tissue contributes to disease progression, we propose neutrophilic TRP channel blockade as a potential therapeutic option.


Assuntos
Granulócitos/metabolismo , Neutrófilos/metabolismo , Canais de Receptores Transientes de Potencial/metabolismo , Animais , Humanos , Microambiente Tumoral/fisiologia
5.
J Gen Physiol ; 148(2): 97-118, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27481712

RESUMO

The voltage-gated proton channel (HV1) is a widely distributed, proton-specific ion channel with unique properties. Since 2006, when genes for HV1 were identified, a vast array of mutations have been generated and characterized. Accessing this potentially useful resource is hindered, however, by the sheer number of mutations and interspecies differences in amino acid numbering. This review organizes all existing information in a logical manner to allow swift identification of studies that have characterized any particular mutation. Although much can be gained from this meta-analysis, important questions about the inner workings of HV1 await future revelation.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/metabolismo , Mutação , Animais , Humanos , Canais Iônicos/genética , Conformação Proteica
6.
FEBS J ; 283(8): 1453-64, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26866814

RESUMO

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.


Assuntos
Membrana Celular/metabolismo , Insetos/metabolismo , Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Potenciais da Membrana/fisiologia , Prótons , Sequência de Aminoácidos , Animais , Eletrofisiologia , Humanos , Insetos/classificação , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
7.
J Gen Physiol ; 146(5): 343-56, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26458876

RESUMO

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.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/química , Sequência de Aminoácidos , Animais , Células COS , Células HEK293 , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Mutação , Triptofano/química , Triptofano/genética
8.
Sci Rep ; 5: 10320, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25955978

RESUMO

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 AspH(0)-H2O(0)-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.


Assuntos
Canais Iônicos/fisiologia , Arginina/química , Arginina/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Humanos , Canais Iônicos/química , Íons , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Prótons , Trítio/metabolismo , Água/metabolismo
9.
Proc Natl Acad Sci U S A ; 111(50): 18078-83, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25425665

RESUMO

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, Thr(29). 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.


Assuntos
Linfócitos B/metabolismo , Neoplasias Hematológicas/imunologia , Canais Iônicos/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Técnicas de Patch-Clamp , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
J Gen Physiol ; 142(6): 625-40, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24218398

RESUMO

Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hHV1), because the proton concentration is >10(6) times lower than that of other cations. Here we use "selectivity filter scanning" to elucidate the molecular requirements for proton-specific conduction in hHV1. Asp(112), in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp(112) by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for "second site suppressor" activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn(2+). Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anion-permeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/química , Simulação de Dinâmica Molecular , Prótons , Sequência de Aminoácidos , Animais , Células COS , Células HEK293 , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Zinco/farmacologia
11.
J Gen Physiol ; 141(4): 445-65, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23530137

RESUMO

The topological similarity of voltage-gated proton channels (H(V)1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether H(V)1s have a similar structure. We present the construction and validation of a homology model of the human H(V)1 (hH(V)1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hH(V)1 by exploiting the homology of hH(V)1 with VSDs of K(+) and Na(+) channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hH(V)1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp(112). Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hH(V)1. Each Arg residue in the S4 helix of hH(V)1 was replaced by His to test accessibility using Zn(2+) as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg(211), the third Arg residue in the S4 helix in hH(V)1, remains accessible to the internal solution and is located near the charge transfer center, Phe(150).


Assuntos
Canais Iônicos/química , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Animais , Células COS , Humanos , Ativação do Canal Iônico , Canais Iônicos/genética , Canais Iônicos/metabolismo , Potenciais da Membrana , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Filogenia , Estrutura Terciária de Proteína , Prótons , Eletricidade Estática
12.
Wiley Interdiscip Rev Membr Transp Signal ; 1(5): 605-620, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23050239

RESUMO

The biophysical properties of the voltage gated proton channel (H(V)1) are the key elements of its physiological function. The voltage gated proton channel is a unique molecule that in contrast to all other ion channels is exclusively selective for protons. Alone among proton channels, it has voltage and time dependent gating like other "classical" ion channels. H(V)1 is furthermore a sensor for the pH in the cell and the surrounding media. Its voltage dependence is strictly coupled to the pH gradient across the membrane. This regulation restricts opening of the channel to specific voltages at any given pH gradient, therefore allowing H(V)1 to perform its physiological task in the tissue it is expressed in. For H(V)1 there is no known blocker. The most potent channel inhibitor is zinc (Zn(2+)) which prevents channel opening. An additional characteristic of H(V)1 is its strong temperature dependence of both gating and conductance. In contrast to single-file water filled pores like the gramicidin channel, H(V)1 exhibits pronounced deuterium effects and temperature effects on conduction, consistent with a different conduction mechanism than other ion channels. These properties may be explained by the recent identification of an aspartate in the pore of H(V)1 that is essential to its proton selectivity.

13.
J Biol Chem ; 287(12): 9376-88, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22291013

RESUMO

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+) chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the H(V)1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.


Assuntos
Regulação Enzimológica da Expressão Gênica , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Espermatozoides/enzimologia , Superóxidos/metabolismo , Linhagem Celular , Humanos , Masculino , Proteínas de Membrana/genética , NADPH Oxidase 5 , NADPH Oxidases/genética , Motilidade Espermática , Espermatozoides/citologia , Espermatozoides/metabolismo
14.
Am J Physiol Cell Physiol ; 302(1): C286-95, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22012327

RESUMO

Reactive oxygen species (ROS) production by human monocytes differs profoundly from that by neutrophils and eosinophils in its dependence on external media glucose. Activated granulocytes produce vast amounts of ROS, even in the absence of glucose. Human peripheral blood monocytes (PBM), in contrast, are suspected not to be able to produce any ROS if glucose is absent from the media. Here we compare ROS production by monocytes and neutrophils, measured electrophysiologically on a single-cell level. Perforated-patch-clamp measurements revealed that electron current appeared after stimulation of PBM with phorbol myristate acetate. Electron current reflects the translocation of electrons through the NADPH oxidase, the main source of ROS production. The electron current was nearly abolished by omitting glucose from the media. Furthermore, in preactivated glucose-deprived cells, electron current appeared immediately with the addition of glucose to the bath. To characterize glucose dependence of PBM further, NADPH oxidase activity was assessed as hydrogen peroxide (H(2)O(2)) production and was recorded fluorometrically. H(2)O(2) production exhibited similar glucose dependence as did electron current. We show fundamental differences in the glucose dependence of ROS in human monocytes compared with human neutrophils.


Assuntos
Elétrons , Glucose/fisiologia , Monócitos/fisiologia , Células Cultivadas , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Monócitos/enzimologia , Monócitos/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
Nature ; 480(7376): 273-7, 2011 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-22020278

RESUMO

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel, H(V)1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, H(V)1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of H(V)1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H(V)1 requires an acidic group at the selectivity filter.


Assuntos
Ácido Aspártico/metabolismo , Ativação do Canal Iônico/genética , Canais Iônicos/química , Canais Iônicos/metabolismo , Prótons , Ácido Aspártico/genética , Condutividade Elétrica , Histidina/genética , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/genética , Soluções Isotônicas/farmacologia , Lisina/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Fases de Leitura Aberta/genética , Concentração Osmolar , Permeabilidade/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Sacarose/farmacologia
16.
Proc Natl Acad Sci U S A ; 108(44): 18162-7, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22006335

RESUMO

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kH(V)1: it is proton-specific and activated by depolarization, its g(H)-V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp(51)) results in loss of proton-specific conduction. Indirect evidence suggests that kH(V)1 is monomeric, unlike other proton channels. Furthermore, kH(V)1 differs from all known proton channels in activating well negative to the Nernst potential for protons, E(H). This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.


Assuntos
Dinoflagelados/fisiologia , Ativação do Canal Iônico , Animais , Dinoflagelados/genética , Mutação , Prótons
17.
Channels (Austin) ; 4(4): 260-5, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20676047

RESUMO

The voltage-gated proton channel exists as a dimer, although each protomer has a separate conduction pathway, and when forced to exist as a monomer, most major functions are retained. However, the proton channel protomers appear to interact during gating. Proton channel dimerization is thought to result mainly from coiled-coil interaction of the intracellular C-termini. Several types of evidence are discussed that suggest that the dimer conformation may not be static, but is dynamic and can sample different orientations. Zn(2+) appears to link the protomers in an orientation from which the channel(s) cannot open. A tandem WT-WT dimer exhibits signs of cooperative gating, indicating that despite the abnormal linkage, the correct orientation for opening can occur. We propose that C-terminal interaction functions mainly to tether the protomers together. Comparison of the properties of monomeric and dimeric proton channels speaks against the hypothesis that enhanced gating reflects monomer-dimer interconversion.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/metabolismo , Basófilos/metabolismo , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Cinética , Potenciais da Membrana , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , Prótons , Relação Estrutura-Atividade , Zinco/metabolismo
18.
J Physiol ; 588(Pt 9): 1435-49, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20231140

RESUMO

Voltage-gated proton channels are strongly inhibited by Zn(2+), which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn(2+). We hypothesize that high-affinity Zn(2+) binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn(2+) effects were weaker in monomeric channels. Mutation of His(193) and His(140) in various combinations and in tandem dimers revealed that channel opening was slowed by Zn(2+) only when at least one His was present in each monomer, suggesting that in wild-type (WT) H(V)1, Zn(2+) binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn(2+) appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer-dimer interconversion occurs during activation of phagocytes appears to be ruled out.


Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/antagonistas & inibidores , Zinco/farmacologia , Linhagem Celular , Dimerização , Eletrofisiologia , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/genética , Canais Iônicos/química , Canais Iônicos/genética , Cinética , Modelos Moleculares , Mutação/fisiologia , Técnicas de Patch-Clamp , Temperatura Ambiente , Transfecção
19.
Nat Immunol ; 11(3): 265-72, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20139987

RESUMO

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.


Assuntos
Linfócitos B/imunologia , Canais Iônicos/imunologia , Espécies Reativas de Oxigênio/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Linfócitos B/enzimologia , Ativação Enzimática/imunologia , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/imunologia , Proteína Oncogênica v-akt/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais , Quinase Syk
20.
J Biol Chem ; 285(8): 5117-21, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20037153

RESUMO

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH(i) regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-delta phosphorylation sites in the human voltage-gated proton channel, H(V)1 (Hvcn1), Thr(29) and Ser(97), both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-delta. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H(V)1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H(V)1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr(29) is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.


Assuntos
Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Leucócitos/metabolismo , Explosão Respiratória/fisiologia , Treonina/metabolismo , Substituição de Aminoácidos , Carcinógenos/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/genética , Maleimidas/farmacologia , Mutação de Sentido Incorreto , NADPH Oxidases/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Explosão Respiratória/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Treonina/genética
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