Phagocytosis of microbial invaders represents a fundamental defense mechanism the innate immune system. The subsequent killing microbes is initiated by respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation crucial because NADPH functions optimally at neutral pH, yet produces enormous quantities protons. We monitored pH(i) individual human...

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 bioluminescent dinoflagellates, where they were thought to trigger flash by activating luciferase. Proton channel genes subsequently identified human, mouse, Ciona intestinalis , but their dinoflagellates remained unconfirmed. We a candidate gene from Karlodinium veneficum cDNA library based on homology with known genes. K. is predatory, nonbioluminescent dinoflagellate that produces toxins...

The effects of low concentrations extracellular ATP on cytosolic Ca 2+ , membrane potential, and transcription IL-6 were studied in monocyte-derived human macrophages. During inflammation or infection many cells secrete ATP. We show here that application 10 μM UTP induces oscillations with a frequency ≈12 min -1 potential. RT-PCR analysis showed expression P2Y 1 2 11 P2X 4 7 receptors, large-conductance ( KCNMA1 KCNMB1–4 ), intermediate-conductance KCNN4 ) -activated K + channels. unchanged...

The two‐pore‐domain potassium channels TASK‐1, TASK‐3 and TASK‐5 possess a conserved C‐terminal motif of five amino acids. Truncation the C‐terminus TASK‐1 strongly reduced currents measured after heterologous expression in Xenopus oocytes or HEK293 cells decreased surface membrane GFP‐tagged channel proteins. Two‐hybrid analysis showed that domain TASK‐5, but not TASK‐4, interacts with isoforms adapter protein 14‐3‐3. A pentapeptide at extreme RRx(S/T)x, was found to be sufficient for weak...

Voltage-gated proton channels are strongly inhibited by Zn(2+), which binds to His residues. However, in a molecular model, the two externally accessible too far apart coordinate Zn(2+). We hypothesize that high-affinity Zn(2+) binding occurs at dimer interface between pairs of residues from both monomers. Consistent with this idea, effects were weaker monomeric channels. Mutation His(193) and His(140) various combinations tandem dimers revealed channel opening was slowed only when least one...

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 facilitating production of ROS. Here we compare the full-length channel HVCN1 L with a shorter protein isoform, S , which lacks first 20 amino acids. Cells display enhanced currents upon stimulation. In addition, internalized to lesser extent interactions receptor, resulting greater...

The topological similarity of voltage-gated proton channels (H(V)1s) to the voltage-sensing domain (VSD) other ion raises central question whether H(V)1s have a similar structure. We present construction and validation homology model human H(V)1 (hH(V)1). Multiple structural alignment was used construct models open (proton-conducting) state hH(V)1 by exploiting with VSDs K(+) Na(+) known three-dimensional comparative assessment stability their VSD templates performed using massively repeated...

Two years ago, genes coding for voltage-gated proton channels in humans, mice and Ciona intestinalis were discovered. Transfection of cDNA encoding the human HVCN1 (H(V)1) or mouse (mVSOP) ortholog into mammalian cells results currents that are extremely similar to native currents, with a subtle, but functionally important, difference. Expressed exhibit high H(+) selectivity, voltage-dependent gating, strong temperature sensitivity, inhibition by Zn(2+), gating kinetics currents. Like...

Eosinophils and other phagocytes use NADPH oxidase to kill bacteria. Proton channels in human eosinophils neutrophils are thought sustain activity, their opening is greatly enhanced by a variety of activators, including phorbol myristate acetate (PMA). In nonphagocytic cells that lack oxidase, no clear effect PMA on proton has been reported. The basophil granulocyte developmentally closely related the eosinophil but nevertheless does not express oxidase. Thus, one might expect stimulating...

Abstract Voltage-gated proton channels, H V 1, trigger bioluminescence in dinoflagellates, enable calcification coccolithophores and play multifarious roles human health. Because the concentration is minuscule, exquisite selectivity for protons over other ions critical to 1 function. The of open channel requires an aspartate near arginine filter (SF), a narrow region that dictates selectivity, but mechanism unknown. Here we use reduced quantum model elucidate how Asp–Arg SF selects excludes...

Somatic mutations are common in cancer, with only a few driving the progression of disease, while most silent passengers. Some may hinder or even reverse cancer progression. The voltage-gated proton channel (HV1) plays key role cellular pH homeostasis and shows increased expression several malignancies. Inhibiting HV1 cells reduces invasion, migration, extrusion, recovery, impacting tumor Focusing on HVCN1, gene coding for human (hHV1), 197 were identified from three databases: 134 missense...

The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable clearing invading pathogens but can also inflict damage to host tissues. It therefore important prevent an overshoot the TLR-induced response where necessary, and here we show that extracellular ATP doing this in human monocytes. Using reverse transcription-PCR, showed monocytes express P2Y1, P2Y2, P2Y4, P2Y11, P2Y13 receptors, as well...

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 also enhance channel gating profoundly, facilitating its roles charge compensation pH(i) regulation. The "enhanced mode" appears reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-delta phosphorylation sites human voltage-gated channel, H(V)1 (Hvcn1),...

Part of the “signature sequence” that defines voltage-gated proton channel (HV1) is a tryptophan residue adjacent to second Arg in S4 transmembrane helix: RxWRxxR, which perfectly conserved all high confidence HV1 genes. Replacing Trp207 human (hHV1) with Ala, Ser, or Phe facilitated gating, accelerating opening by 100-fold, and closing 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating WT Trp favors closed state. The Arrhenius activation...

Significance A large family of membrane proteins, voltage-gated ion channels, regulate a vast array physiological functions in essentially all life forms. How these molecules sense potential and respond by creating ionic conduction is incompletely understood. The voltage sensors channels contain “hydrophobic gasket,” ring hydrophobic amino acids near the center membrane, separating internal external aqueous solutions. Although proton H V 1, resemble voltage-sensing domains other they differ...

The voltage-gated proton channel, HV 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the halfway across membrane. To distinguish between both mobility putative case estimated. predicted single-channel permeability 2.3 × 10-12 cm3 s-1 reflects permeability-governing number hydrogen bonds molecules in single-file configuration and pore residues. However,...

The potassium channels TASK-1 and TASK-3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression HEK293 cells showed that the single-channel conductance of increased approximately four-fold after removal external cations, whereas was unaffected. Replacing glutamate at position 70 by a lysine or arginine residue abolished reverse mutation (K70E) induced organic polycations spermine ruthenium red modulated similar way as Ca2+ Mg2+....

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. Although these molecules distinct entities, with no proven physical interaction, their presence activity many cells appears be coordinated. We describe interactions discuss several types of mechanisms that might explain them.

Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hHV1), because concentration >106 times lower than that of other cations. Here we use “selectivity filter scanning” elucidate molecular requirements for proton-specific conduction in hHV1. Asp112, middle S1 transmembrane helix, an essential part wild-type (WT) channels. After neutralizing Asp112 by mutating it Ala (D112A), introduced Asp at each position along...

Reactive oxygen species (ROS) production by human monocytes differs profoundly from that neutrophils and eosinophils in its dependence on external media glucose. Activated granulocytes produce vast amounts of ROS, even the absence Human peripheral blood (PBM), contrast, are suspected not to be able any ROS if glucose is absent media. Here we compare neutrophils, measured electrophysiologically a single-cell level. Perforated-patch-clamp measurements revealed electron current appeared after...

Abstract The biophysical properties of the voltage‐gated proton channel (H V 1) are key elements its physiological function. is a unique molecule that in contrast to all other ion channels exclusively selective for protons. Alone among channels, it has voltage‐ and time‐dependent gating like ‘classical’ channels. H 1 furthermore sensor pH cell surrounding media. Its voltage dependence strictly coupled gradient across membrane. This regulation restricts opening specific voltages at any given...