With a single gene encoding H
1 channel, proton channel diversity is particularly low in mammals compared to other members of the superfamily of voltage-gated ion channels. Nonetheless, mammalian H
1 ...channels are expressed in many different tissues and cell types where they exert various functions. In the first part of this review, we regard novel aspects of the functional expression of H
1 channels in mammals by differentially comparing their involvement in (1) close conjunction with the NADPH oxidase complex responsible for the respiratory burst of phagocytes, and (2) in respiratory burst independent functions such as pH homeostasis or acid extrusion. In the second part, we dissect expression of H
channels within the eukaryotic tree of life, revealing the immense diversity of the channel in other phylae, such as mollusks or dinoflagellates, where several genes encoding H
channels can be found within a single species. In the last part, a comprehensive overview of the biophysical properties of a set of twenty different H
channels characterized electrophysiologically, from Mammalia to unicellular protists, is given.
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.
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.
The respiratory burst of phagocytes is essential for human survival. Innate immune defence against pathogens relies strongly on reactive oxygen species (ROS) production by the NADPH oxidase (NOX2). ...ROS kill pathogens while the translocation of electrons across the plasma membrane via NOX2 depolarizes the cell. Simultaneously, protons are released into the cytosol. Here, we compare freshly isolated human polymorphonuclear leukocytes (PMN) to the granulocytes-like cell line PLB 985. We are recording ROS production while inhibiting the charge compensating and pH regulating voltage-gated proton channel (HV1). The data suggests that human PMN and the PLB 985 generate ROS via a general mechanism, consistent of NOX2 and HV1. Additionally, we advanced a mathematical model based on the biophysical properties of NOX2 and HV1. Our results strongly suggest the essential interconnection of HV1 and NOX2 during the respiratory burst of phagocytes. Zinc chelation during the time course of the experiments postulates that zinc leads to an irreversible termination of the respiratory burst over time. Flow cytometry shows cell death triggered by high zinc concentrations and PMA. Our data might help to elucidate the complex interaction of proteins during the respiratory burst and contribute to decipher its termination.
Voltage‐gated proton channels (HV1) are expressed in eukaryotes, including basal hexapods and polyneopteran insects. However, currently, there is little known about HV1 channels in insects. A ...characteristic aspartate (Asp) that functions as the proton selectivity filter (SF) and the RxWRxxR voltage‐sensor motif are conserved structural elements in HV1 channels. By analysing Transcriptome Shotgun Assembly (TSA) databases, we found 33 polyneopteran species meeting these structural requirements. Unexpectedly, an unusual natural variation Asp to glutamate (Glu) at SF was found in Phasmatodea and Mantophasmatodea. Additionally, we analysed the expression and function of HV1 in the phasmatodean stick insect Extatosoma tiaratum (Et). EtHV1 is strongly expressed in nervous tissue and shows pronounced inward proton conduction. This is the first study of a natural occurring Glu within the SF of a functional HV1 and might be instrumental in uncovering the physiological function of HV1 in insects.
We report the discovery of voltage‐gated proton channels (HV1) in polyneopteran insects. We found a novel, naturally occurring glutamate in the selectivity filter of HV1. We further characterized the HV1 of the phasmatodean Extatosoma tiaratum, EtHV1. This study provides the first step in the discovery of the physiological function of HV1 channels in insects and its relationship with their evolution.
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.
Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which ...nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pHi in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na⁺/H⁺ antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pHi decreased sharply, and recovery required both Na⁺/H⁺ antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn²⁺ resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn²⁺. Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis.
The voltage‐gated proton channel 1 (HV1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV1 ...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 HV1 channel. In the present study, we report the existence of an HV1‐type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV1 orthologues in various polyneopteran insects. To confirm that these putative HV1 orthologues were functional channels, we studied the HV1 channel of Nicoletia phytophila (NpHV1), an insect of the Zygentoma order, in more detail. NpHV1 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, NpHV1 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.
The HV1 voltage‐gated proton channel extrudes cellular protons and compensates charge during the respiratory burst. In contrast to a postulated dogma, we report the existence of HV1 in insects. NpHV1 from Nicoletia phytophila is proton selectivity, and shows pH‐ and voltage‐dependent gating. Substitutions of an aspartate in S1 leads to a loss of proton‐selective conduction, confirming its importance as selectivity filter in all HV1s.
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 (HVCN1 S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1 S 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 HVCN1 L, Thr ²⁹. Furthermore, the association of HVCN1 S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1 S 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 HVCN1 S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.
Significance B lymphocytes are crucial cells in immune responses. Their activity is regulated by signaling pathways involving reactive oxygen species (ROS). Voltage-gated proton channels modulate B-cell responses by facilitating production of ROS. Here we compare the full-length proton channel HVCN1 L with a shorter protein isoform, HVCN1 S, which lacks the first 20 amino acids. Cells with HVCN1 S display enhanced proton currents upon stimulation. In addition, HVCN1 S is internalized to a lesser extent by interactions with the B-cell receptor, resulting in greater plasma membrane expression. Finally, HVCN1 S expression results in greater proliferation and migration. Compared with normal B lymphocytes, HVCN1 S expression is higher in B-cell lines and in B cells from patients with chronic lymphocytic leukemia, where it may contribute to disease pathogenesis.
Voltage-gated proton channel in a dinoflagellate Smith, Susan M. E; Morgan, Deri; Musset, Boris ...
Proceedings of the National Academy of Sciences - PNAS,
11/2011, Letnik:
108, Številka:
44
Journal Article
Recenzirano
Odprti dostop
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, kHV1: it is proton-specific and activated by depolarization, its gH–V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. 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.
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