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

Abstract

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

Figure 1

Topology of three molecules that contain voltage-sensing domains (VSD): a generic K⁺ channel, the proton channel, and a voltage-sensitive phosphatase. The upper row shows the orientation of a monomer in the membrane, with the transmembrane helical segments labeled S1, S2, etc. The lower row shows the assembled tetramer, dimer, or monomer, respectively. Ion conduction in the K⁺ channel occurs through the single central pore (in red) which is comprised of four S5–S6 pairs. H_V1 is a dimer in mammals^– but each monomer has an intrinsic conduction pathway; monomeric constructs retain the major functional properties of the dimer^,–. S1–S4 form the VSD that senses membrane potential, which is transduced into channel opening or closing (gating) in “voltage-gated” ion channels, or into changes in the rate of phosphatase activity in the voltage sensing phosphatase (VSP). Reprinted with permission from. Copyright 2010, American Physiological Society.

Figure 2

Fluorescence-based H⁺ flux assay for vesicles containing hH_V1 (dark blue), the VSD of KvAP channels (green), KvAP (dark green), the paddle chimera (cyan), and empty vesicles (red). Valinomycin and protonophore CCCP were added at the indicated time points. Vesicles loaded with K⁺ were placed in a low K⁺ buffer. Valinomycin allows K⁺ efflux which drives H⁺ influx, detected by a fluorescent dye. Reprinted with permission from. ©James Anthony Letts, 2014.

Figure 3

A maximum likelihood phylogenetic tree from a multiple sequence alignment of the VSD portion of 37 H_V1s. Branch lengths are proportional to the distance between sequences. Eight genes confirmed to be H_V1 by direct electrophysiological measurement are starred; the rest are predicted to be H_V1. Two more genes not shown have been confirmed recently by voltage-clamp, from a bioluminescent dinoflagellate Lingulodinium polyedrum, and Nicoletia phytophila [personal communication from G. Chaves, C. Derst, B. Musset]. Reprinted with permission from. ©2011 by The National Academy of Sciences of the USA.

Figure 4

Transmembrane domain of the human voltage gated proton channel, hH_V1. The four helical segments are color-coded (S1 red, S2 yellow, S3 green, S4 blue); the extracellular end is at the top. Acidic and basic residues are labeled; dashed lines indicate salt bridges predicted in molecular dynamics simulations of a homology model to stabilize the open channel. D112 is crucial to proton selectivity. ©. Originally published in the Journal of General Physiology. doi: 10.1085/jgp.201210856

Figure 5

Unrooted phylogram of 122 VSDs from several classes of VSD-containing molecules. The branch length shows the degree of difference from neighbors. Despite including only the VSD (S1–S4) and excluding the pore-forming S5–S6 domain, all K⁺ channels separated onto one branch, Na⁺ and Ca²⁺ channels onto another branch. A third branch includes voltage-sensing phosphatases, H_V1, and c15orf27, molecules of unknown function. Reprinted with permission from. ©2011, Nature Publishing Group.

Figure 6

The Asp residue that is crucial to proton selectivity in hH_V1 can be shifted from position 112 to 116 in the S1 helix. The diagrams show S1 and S4 segments schematically in the open hH_V1 channel. The three S4 Arg residues (R1-R3 in blue) are numbered from the outside (R205, R208, and R211). Yellow indicates Val, red Asp, and gray all positions where Asp did not produce functioning channels. The families of voltage-clamp currents (in 10 mV increments up to the voltage indicated) above each diagram illustrate H⁺-selective currents in WT, no current in D112V, and H⁺-selective currents in the D112V/V116D double mutant, all at pH_o 7.0 and pH_i 5.5. ©. Originally published in the Journal of General Physiology. doi: 10.1085/jgp.201311045

Figure 7

Proton selectivity occurs because H₃O⁺ is uniquely able to break the Asp-Arg connection in the selectivity filter, opening its own conduciton pathway. Quantum calculations reveal that Asp and Arg interact in the hH_V1 selectivity filter via two hydrogen bonds, with stable optimized configurations with Asp (top left) or Arg (top right) protonated. Introducing a hydronium ion, H₃O⁺ into either (lower row) results in protonation of Asp producing a neutral water molecule that mediates interactions between side chains. Computed G values are negative, indicating a favorable forward reaction, assuming dielectric constants of either 4 or 30 to reflect low or high solvent accessibility. Figure generously provided by Karin Mazmanian.

Figure 8

Predicted aqueous accessibility (blue surface) of the pore of the closed mouse H_V1 channel, based on its crystal structure. Dashed lines show membrane surface; the top is the extracellular end. [From]

Figure 9

Maximum conductance of hH_V1 mutants in which Asp has been replaced by the indicated amino acid. Acidic, basic, or neutral side chains are indicated by red, aqua, or blue colors. The more hydrophobic the side chain, the smaller the conductance. Hydrophobicity, defined as in, increases to the left. Data are from or unpublished data from the same authors.

Figure 10

The voltage at which channels open is determined by the pH gradient, pH. (A) Families of proton currents recorded in rat alveolar epithelial cells at several pH, indicated as pH_o//pH_i, during depolarizing voltage pulses shown in 20 mV increments. The peak currents from these families are plotted in B, with diamonds for pH_i 5.5, squares for pH_i 6.5, and triangles for pH_i 7.5. @. Originally published in the Journal of General Physiology. doi: 10.1085/jgp.105.6.861

Figure 11

Proton channels trigger the flash in bioluminescent dinoflagellates. The diagram on the left and the cartoon on the right illustrate J.W. Hastings’ original proposal. Movement of water initiates an action potential that travels along the vacuolar membrane (the tonoplast) and invades the scintillon membrane where it opens voltage gated proton channels. H⁺ readily enters the luciferase-containing scintillon due to the enormous driving force; the vacuole pH is 3.5–4.5^,. The resulting influx of H⁺ activates the acid-activated luciferase^, contained in the scintillon, as well as releasing luciferin from its binding protein. Both events enable the enzyme luciferase to catalyze the oxidation of its substrate luciferin to an excited species that releases a photon, resulting in the flash. Reprinted with permission from. ©2001 by Elsevier. The first gene for a dinoflagellate proton channel (or ion channel of any kind) was obtained from a cDNA library from Karlodinium veneficum. When expressed in mammalian cells the gene product (kH_V1) produced depolarization-activated currents (A) that reversed near the Nernst potential for H⁺ (E_H) and were thus proton selective (C). Unlike any other proton channel identified to date, kH_V1 opens well below E_H and thus conducts inward current, as evident in the current-voltage curve in B. This property is ideally suited to the proposed purpose, because in the tonoplast, H_V1 would orient with the vacuole being topologically extracellular. H_V1 from any other species would prefer to extrude acid. To trigger the flash, H_V1 must open and allow inward current, precisely as observed. In addition to enabling H⁺ flux into the scintillon, these proton channels may also mediate the action potential that triggers the flash. Reprinted with permission from. ©2011 by The National Academy of Sciences of the USA. Artwork by Zina Deretsky, National Science Foundation.

Figure 12

A symbiotic relationship exists between H_V1 and NOX2, the phagocyte NADPH oxidase enzyme complex. When a bacterium is engulfed into the nascent phagosome, NOX2 rapidly assembles in the phagosome membrane (the membrane is depicted as a tan double line) and becomes extremely active, producing a “respiratory burst.” This enzyme produces bactericidal reactive oxygen species (ROS) by extracting an electron from NADPH inside the cell, and transferring it across a redox chain to reduce extracellular oxygen to superoxide anion, from which other ROS are produced. Because NOX2 moves electrons across the membrane it is electrogenic and tends to depolarize the phagosome membrane, while leaving behind a proton in the cell tends to acidify the cytoplasm. H_V1 in phagocytes^,– acts as a countercharge, limiting depolarization^,,–. At the same time, H⁺ efflux through H_V1 restores cytoplasmic pH. Two other consequences of H_V1 activity during the “respiratory burst” are to limit the osmotic consequences of charge compensation and to provide the necessary substrate for ROS production in the phagosomes. Roughly 95% of the charge compensation required is provided by H_V1, and ROS production is attenuated by up to 75% in H_V1 knock-out mice^,,–,, or rats. Reprinted with permission from. Copyright 2010, American Physiological Society.

Figure 13

Currents in 10-mV increments up to 80 mV before (A) and after (B) PMA (phorbol myristate acetate) stimulation. Phosphorylation of H_V1 by the PKC activator, PMA results in faster activation, slower deactivation, larger currents, and activation at more negative voltages. All changes increase the activity and response of H_V1. Reprinted with permission from. ©2008 by The National Academy of Sciences of the USA.

Figure 14

Enhanced gating response of hH_V1 in a B lymphocyte from a patient with chronic lymphocytic leukemia. Control is the g_H derived from a family of proton currents in a resting cell. After PMA stimulation, the current amplitude increased, activation (turn-on) became faster, and as evident in the graph, the g_H turned on at voltages at least 20 mV more negative. The PKC inhibitor, GF109203X (GFX), reversed all of these changes, confirming they were due to phosphorylation of the channel. Reprinted with permission from. ©2014 by The National Academy of Sciences of the USA.

Figure 15

Weak base inhibitors are weak inhibitors of hH_V1. Families of currents during identical 12-s pulses in 10-mV increments up to +100 mV from an inside-out patch of membrane, in the absence or presence of 1 mM chlorpromazine (CPZ). The cationic form of CPZ was proposed to inhibit from the internal solution. Unpublished data by V.V. Cherny and T.E. DeCoursey.

Figure 16

Complex interaction of 400 nM 2GBI with the F150A mutant of hH_V1 is seen in dimer (A), but not in monomer (B). Pulse pairs to +140 mV were applied separated by a variable interval at −80 mV. Current decay during the pulses reflects block by internally-applied drug, with the time course of recovery reflected in the peak current during the second pulse of the pair. From doi.org/10.1016/j.neuron.2012.11.013 ©2013 by Elsevier user license.