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Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family - PubMed

Review

Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family

Thomas E DeCoursey. Physiol Rev. 2013 Apr.

Abstract

Voltage-gated proton channels (H(V)) are unique, in part because the ion they conduct is unique. H(V) channels are perfectly selective for protons and have a very small unitary conductance, both arguably manifestations of the extremely low H(+) concentration in physiological solutions. They open with membrane depolarization, but their voltage dependence is strongly regulated by the pH gradient across the membrane (ΔpH), with the result that in most species they normally conduct only outward current. The H(V) channel protein is strikingly similar to the voltage-sensing domain (VSD, the first four membrane-spanning segments) of voltage-gated K(+) and Na(+) channels. In higher species, H(V) channels exist as dimers in which each protomer has its own conduction pathway, yet gating is cooperative. H(V) channels are phylogenetically diverse, distributed from humans to unicellular marine life, and perhaps even plants. Correspondingly, H(V) functions vary widely as well, from promoting calcification in coccolithophores and triggering bioluminescent flashes in dinoflagellates to facilitating killing bacteria, airway pH regulation, basophil histamine release, sperm maturation, and B lymphocyte responses in humans. Recent evidence that hH(V)1 may exacerbate breast cancer metastasis and cerebral damage from ischemic stroke highlights the rapidly expanding recognition of the clinical importance of hH(V)1.

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Figures

Figure 1.
Figure 1.

The hydrogen bonded chain (HBC) mechanism for proton conduction through proteins. Shown are hydroxyl groups forming a continuous HBC. A: each proton hops one position along the chain, with the final group releasing a proton into the distal solution. B: after the transfer, the chain is not able to accept another proton from the proximal side until all the groups have reoriented. Permeation of OH− occurs when a proton leaves the distal end of the HBC (neutralizing OH− to form H2O), and the defect migrates by retrograde H+ hopping. [Redrawn from Nagle and Morowitz (365).]

Figure 2.
Figure 2.

Phylogenetic tree of 37 known and predicted proton channels. Branch length indicates the evolutionary distance between sequences. This maximum likelihood phylogenetic tree was constructed from a multiple sequence alignment of the voltage sensing domain (VSD) portion of 37 HV1s using 100 bootstraps. The eight HV1 genes that have been confirmed by heterologous expression and electrophysiological recording are starred. Bootstrap values >60 are shown; these numbers indicate the probability that the branch is genuine. [From Smith et al. (462).]

Figure 3.
Figure 3.

Architectural features of K+ channels, H+ channels, and voltage-sensing phosphatases (VSP). The top row shows the monomers from which the final oligomer (bottom row) is constructed. K+ channels are tetramers of subunits that each contain six membrane-spanning regions, of which S1-S4 comprise the voltage sensing domain (VSD) and S5-S6 form the pore. S5-S6 regions from each subunit together form a single central pore, which is surrounded by four VSDs (bottom panel). HV1 contains S1-S4 regions that are quite similar to the K+ channel VSD, but lacks a pore domain (416, 432). The proton channel is a dimer held together by coiled-coil interactions in the C-terminal domain, in which each monomer has its own conduction pathway (259, 286, 497). Phosphorylation of Thr29 in the intracellular N terminus converts the channel from “resting mode” to “enhanced gating mode” (117, 335, 352). The VSP has similar S1-S4 regions but lacks conduction. It senses membrane potential and modulates phosphatase enzyme activity accordingly (348, 349). [From DeCoursey (105).]

Figure 4.
Figure 4.

Phylogenetic relationship between the proton channel (HV1) family and other VSD-containing proteins. Unrooted phylogram from a maximum likelihood analysis of 122 VSDs shows that HV1 sequences appear on a branch distinct from other VSDs. The phylogenetic analysis was performed on VSD sequences only and did not include the cation channel pores or N and C termini. Sequences are color coded: Kv, voltage-gated K+ channel; Nav, voltage-gated Na+ channel; Cav, voltage-gated Ca2+ channel; VSP, voltage-sensitive phosphatase; C15orf27, protein of unknown function. Branches with likelihood support values (a measure of confidence in a branch's appearance in a tree) <0.50 are collapsed. [From Musset et al. (362).]

Figure 5.
Figure 5.

Sequence logos derived from alignment of the primary sequences of the four TM segments of the Karlodinium proton channel, kHV1 (top row) with the TM segments of 37 members of the HV1 family, 15 of C15orf27, 15 of VSP, and 13 KV channels. The letter height (single letter amino acid abbreviations) in the sequence logos shows the proportion of all sequences with a particular amino acid at that position. The overall height of the stack indicates information content (439) or sequence conservation at that position (88). The mean (± SD) numbers of amino acids in the N and C termini are given to the left and right, respectively. The examples numbered for each family are hHV1, human C15orf27, CiVSP, and Shaker for KV. [From Smith et al. (462).]

Figure 6.
Figure 6.

Photobleaching of GFP-tagged hHV1 occurs in two distinct irreversible steps, indicating that the channel exists mainly as a dimer. A: blue circles indicate individual channels that were followed over time. B: time course of fluorescence intensity of one spot (i.e., channel). C: illustrates that most channels bleached in two steps. [From Tombola et al. (497), with permission from Elsevier.]

Figure 7.
Figure 7.

Possible dimer interfaces for hHV1. A: cross-linking between Cys residues introduced at several positions on S1 (red) led to this dimer model (286). B: competition between H+ and Zn2+ for a high-affinity binding site led to an alternative model. Apposition of S2 (yellow) and S3 (green) segments allows the Zn2+ binding residues His140 and His193 to approach each other closely enough to form potential bidentate Zn2+ binding sites. A recent study proposed that the C terminus and the S4 helix form a single rigid helix through which the C termini mediate cooperative gating and that any direct interaction within the TM region is inconsequential to this process (172). [From Musset et al. (359).]

Figure 8.
Figure 8.

Molecular anatomy of the M2 proton channel transmembrane domains. Three of the four monomers that form the channel are shown as gray ribbons. The tetrad of His37 residues are orange; red spheres are water molecules. The “entry cluster” comprises six waters, four of which are hydrogen bonded to the His37. The two waters in the “bridging cluster” are also hydrogen bonded to the His. The permeating proton is thought to be delocalized among the His box and associated water clusters. Black lines indicate hydrogen bonds. [From Acharya et al. (2).]

Figure 9.
Figure 9.

Ionic strength dilution with isotonic sucrose reveals that the D112A/D185M mutation of hHV1 results in anion selectivity. Tail current reversal potential measurements are shown at several levels of dilution of the bath solution. Bath and pipette solutions contained ∼130 mM TMA+ CH3SO3− at pH 7.0 and 5.5, respectively. The control Vrev (A) is far from EH, which is −87 mV, but not significantly different from that in D112A mutants (362). Dilution of the external solution shifts Vrev progressively positively. [Unpublished data by Deri Morgan, from a study reported in Musset et al. (362).]

Figure 10.
Figure 10.

Identification of the selectivity filter of hHV1. The replacement of Asp112 by serine converts the human proton channel hHV1 into an anion channel. Tail current measurements at symmetrical pH 5.5 with ∼130 mM TMA+ CH3SO3− in pipette and bath (top) and after bath replacement with TMA+ Cl− (bottom) reveal that Vrev shifted nearly −50 mV, indicating higher permeability to Cl− than to CH3SO3−. In each, a 4-s depolarizing prepulse activated the conductance, then the voltage was stepped back to a range of voltages in 10-mV increments to determine the zero current potential, Vrev. [Unpublished figure by Deri Morgan, from a study reported in Musset et al. (362).]

Figure 11.
Figure 11.

Guanidinium (Gu+) is not an effective blocker of hHV1. Families of currents in COS-7 cells expressing R211A (A) and wild-type hHV1 channels (B) during pulses applied in 10-mV increments up to +50 mV from a holding potential of −40 mV. Both cells were studied in whole cell configuration in symmetrical solutions containing 100 mM GuCl at pH 8.0. (Unpublished data by Vladimir V. Cherny.)

Figure 12.
Figure 12.

Accessibility of S4 residues in open and closed HV1 channels. Cys scanning with MTS reagents was done in CiHV1 (185). Residues in CiHV1 that were externally accessible preferentially when the channel was open (i.e., measured at positive voltage) are orange; residues that were internally accessible preferentially when the channel was closed (i.e., measured at negative voltage) are green; residues not accessible in either state are red. Yellow shading indicates state-dependent accessibility; gray complete inaccessibility. Minimum stretches that are inaccessible in closed or open states deduced from the CiHV1 MTS data are indicated by arrows. For mHV1, presumably in the closed state, PEGylation assays indicated residues with full accessibility (blue), partial accessibility (brown), and inaccessibility (red), with shading indicated partial (gray) or complete (dark gray) inaccessibility (428). For hHV1, Zn2+ inhibition of currents produced by Arg→His mutants indicated external accessibility (orange) and internal accessibility (blue) of open channels (361).

Figure 13.
Figure 13.

Dimeric (WT) hHV1 proton currents activate sigmoidally (A), whereas monomeric channels activate more rapidly and exponentially (B). [From Musset et al. (360).] C: the fluorescence signal from a tag on S4 of CiHV1 (red) reports movement of S4 during depolarization to the indicated voltages and changes faster and earlier than the current turns on (black line), which exhibits sigmoid activation kinetics. The S4 signal squared (green) mimics the current. [From Gonzalez et al. (185), with permission from The Nature Publishing Group.]

Figure 14.
Figure 14.

All proton channels share ΔpH-dependent gating. When Vthreshold is determined along with Vrev over a range of pH gradients, the result falls on a line defined by Eqs. 4 and 5 (text). Combined data from 15 different cells types were described by the blue line (Eq. 5, using values given in TABLE 3). The red dashed line shows equality between Vrev and Vthreshold, a data point that falls above this line means that when the channel opens, only outward current is possible. These channels open only when the electrochemical gradient is outward, when opening will result in acid extrusion from the cell. Data from the dinoflagellate, K. veneficum, have an identical slope, but are offset by 60 mV to more negative values. As a result, this channel opens and conducts inward H+ current over a wide voltage range, and consequently must serve quite different functions. [Redrawn and combined from DeCoursey (104) and Smith et al. (462).]

Figure 15.
Figure 15.

Strong pHo dependence of the inhibitory effects of Zn2+ reflects competition for an external binding site. Each row shows currents in a rat alveolar epithelial cell during three identical voltage-clamp families, with pulses applied in 10-mV increments, in the absence or presence of Zn2+. The two main effects, the slowing of activation and the shift of the gH-V relationship, both require dramatically higher [Zn2+] at low pHo. [From Cherny and DeCoursey (71).]

Figure 16.
Figure 16.

Evidence that Zn2+ binds with high affinity at the interface between the two monomers in the dimeric human proton channel. The Zn2+ dependence of the slowing of channel opening (τact) by Zn2+ is plotted for WT hHV1 and six related constructs in which one or both of the key His residues, His140 and His193, were replaced with Ala, including three tandem dimers. Inset cartoons show His as solid, Ala as open, with circles for position 140 and squares for position 193. Slowing occurs only when at least one His is present in each monomer. [From Musset et al. (360).]

Figure 17.
Figure 17.

Proton channels trigger a light flash in bioluminescent dinoflagellates in response to mechanical stimulation. An action potential travels along the tonoplast, the membrane surrounding the large central flotation vacuole, and invades the scintillon, a small organelle that contains high concentrations of luciferin, luciferase, and luciferin binding protein. The depolarization opens voltage-gated proton channels in the scintillon membrane. The resulting proton influx from the vacuole (at pH 3.5–4.5; Refs. 374, 380) directly triggers the flash both by activating luciferase and by causing LBP to release luciferin, the substrate for luciferase. A proton channel with properties like those in kHV1 (FIGURES 18 AND 19) could also mediate the action potential. [From Hastings (202).]

Figure 18.
Figure 18.

The proton channel kHV1, from the dinoflagellate Karlodinium veneficum, exhibits inward proton currents. A: during depolarizing pulses in 5-mV increments from Vhold = −60 mV through +15 mV at symmetrical pH 7.0, there are inward currents over a wide voltage range. B: the current-voltage curve resembles that of voltage-gated Na+ or Ca2+ channels, which are also capable of mediating action potentials. [From Smith et al. (462).]

Figure 19.
Figure 19.

Proton channels from the dinoflagellate Karlodinium veneficum differ from all known HV1 in producing distinct inward currents, but share ΔpH-dependent gating. Because the gH-V relationship is shifted ∼60 mV more negative than in other species at any given ΔpH, Vthreshold occurs well negative to EH. A–C show families of proton currents in an inside-out patch at pHo 7.0 and at the indicated pHi. Pulses were applied in 10 mV increments with the most positive voltage labeled. The corresponding current-voltage relationships (D) resemble those for voltage-gated Na+ channels, especially at low pHi. The gH-V relationship is (E) shifted by changes in pHi, as in other proton channels. [From Smith et al. (462).]

Figure 20.
Figure 20.

Role of proton channels in calcification and pH homeostasis in coccolithophores. Mature coccoliths arranged on the extracellular surface surround the cell to form a coccosphere (A). Coccolith formation occurs within an intracellular Golgi-derived coccolith vesicle (B). Calcium carbonate (CaCO3) precipitation requires the production of carbonate (CO32−) from bicarbonate (HCO3−) and results in net production of H+. H+ must be rapidly removed from the coccolith vesicle to maintain a pH conducive to CaCO3 precipitation. Once in the cytosol (C), some H+ may be utilized by photosynthesis in the production of CO2 from HCO3− (see text); however, H+ efflux provides an efficient mechanism to prevent cytosolic acidosis, which inhibits calcification (489). Patch-clamp studies indicate that Cl− and H+ are the dominant transmembrane conductances in coccolithophores (487, 489). At normal seawater pH 8.2, pHi of 7.0–7.2 and membrane potential approximately −60 mV maintained by a Cl− inward rectifier (D) (487), a drop in pHi or membrane depolarization or both would activate plasma membrane H+ channels (E), providing an extremely efficient and rapid mechanism for maintaining constant intracellular pH. [From Taylor et al. (489).]

Figure 21.
Figure 21.

Regulation of the pH of airway surface liquid (ASL) by the combined reciprocal efforts of hHV1 and CFTR. The anion transporter CFTR extrudes HCO3− passively, alkalinizing the ASL. This increased pHo opens proton channels, which passively extrude H+, restoring the ASL pH to its normal value. [From Fischer (156), with permission from Wiley.]

Figure 22.
Figure 22.

Involvement of hHV1 in B lymphocyte signaling pathways. Antigen binding to the BCR results in phosphorylation of internal signaling molecules, with negative feedback from CD22. BCR stimulation results in NADPH oxidase activation, producing H2O2 that diffuses back into the cell where it inhibits SHP-1, thereby relieving inhibition of BCR signaling by the latter. See References and for more detailed descriptions. [From Capasso et al. (62), with permission from Elsevier.]

Figure 23.
Figure 23.

The motility of human spermatozoa requires functional interaction between NOX5 and hHV1. H2O2 induces calcium influx and the activation of c-Abl through tyrosine phosphorylation, both of which contribute to NOX5 activation. The generation of superoxide anion results in production of protons in the cytoplasm, which are extruded via the proton channel to maintain the optimal pH for NOX5 activity. Inhibition of this pathway abrogates the stimulatory effect of H2O2 on spermatozoa motility (356). [From Musset et al. (356), with permission from The American Society for Biochemistry and Molecular Biology.]

Figure 24.
Figure 24.

Molecules and transporters that participate in charge compensation and pH regulation during the respiratory burst. A phagocyte is shown engulfing a bacterium into a nascent phagosome, which will close and become an intracellular compartment. NADPH oxidase assembles preferentially in the phagosomal membrane in neutrophils (44, 246, 389) and begins to function before the phagocytic cup has sealed (297). The oxidase assembles mainly at the plasma membrane in eosinophils (273), macrophages (246), and neutrophils that are stimulated with soluble agonists (41, 390). NADPH oxidase activity drives the entire system. Electrons from intracellular NADPH are translocated across the membrane to reduce O2 to superoxide anion (O2−) in the phagosome or extracellular space. This electrogenic process (209) can be measured directly as electron current (117, 120, 442). Each electron removed from the cell leaves behind approximately one proton. Thus NADPH oxidase activity tends to depolarize the membrane, decrease pHi, and increase pHo or pHphagosome. ClC-3 is shown moving H+ into the phagosome in exchange for Cl−, as might occur at depolarized potentials that exist during the respiratory burst (275). In endosomes lacking NADPH oxidase, ClC-3 is thought to operate in the reverse direction, removing H+ and injecting Cl− into the interior to compensate for electrogenic H+-ATPase activity (240). HOCl is membrane permeant (506) and reacts rapidly with cytoplasmic contents such as taurine (322) or glutathione (519). As a result, HOCl effectively shuttles protons out of the phagosome (334). Despite the arrows, the protons in any compartment are equivalent. [From DeCoursey (105).]

Figure 25.
Figure 25.

Intracellular pH (pHi) during phagocytosis in four human neutrophils, imaged with SNARF-1 using the SEER approach (281). Pseudocolor images of the cells are positioned near the pHi record at the corresponding time. When the opsonized zymosan particle is engulfed, there is a rapid spike of acidification, followed in most (A), but not all cells (B) by rapid recovery. The Na+/H+ antiport inhibitor dimethylamiloride (DMA) consistently prevented recovery (C) as did the proton channel inhibitor Zn2+ (D). The relative impact on pHi was hHV1 > Na+/H+ antiport > H+-ATPase. [From Morgan et al. (334).]

Figure 26.
Figure 26.

Proton currents in a human neutrophil in perforated-patch configuration at rest (A) and in the enhanced gating mode (B). Identical families of depolarizing pulses were applied to −20 through +80 mV in 10-mV increments from a holding potential of −40 mV, at symmetrical pH 7.0. After stimulation by PMA, proton currents activate faster and deactivate more slowly, the maximum conductance is increased, and the voltage at which channels first open, Vthreshold, is shifted by −40 mV. [From DeCoursey et al. (117).]

Figure 27.
Figure 27.

Regulation of enhanced gating of the proton channel, as well as NADPH oxidase activity, by PKC-dependent phosphorylation. Top left: proton currents during test pulses to +60 mV applied every 30 s from a holding potential of −60 mV. Examples of currents at an expanded time base are superimposed (right) with lowercase letters identifying the time of the pulse. At the arrows, the PKC activator PMA or the PKC inhibitor GFX (GF109203X) were added. Bottom left: the current at −60 mV at high gain (with pulses blanked) shows inward electron current that reflects NADPH oxidase activity. [From Morgan et al. (335).]

Figure 28.
Figure 28.

The naturally occurring hHV1 mutation M91T shifts Vthreshold of the expressed channel by ∼30 mV to more positive voltages. The slopes of the fitted lines, as defined by Eq. 5, are the same, but the offset voltage is +2 mV for WT, and +34 mV for M91T. The mutant channel requires a larger ΔpH to open. [From Iovannisci et al. (233).]

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