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Voltage‐Gated Proton Channels

Thomas E. DeCoursey

10.1002/cphy.c100071

Source: Volume 2, Issue 2, April 2012

Published online: April 2012

Full Article on Wiley Online Library



Abstract

Voltage‐gated proton channels, HV1, have vaulted from the realm of the esoteric into the forefront of a central question facing ion channel biophysicists, namely, the mechanism by which voltage‐dependent gating occurs. This transformation is the result of several factors. Identification of the gene in 2006 revealed that proton channels are homologues of the voltage‐sensing domain of most other voltage‐gated ion channels. Unique, or at least eccentric, properties of proton channels include dimeric architecture with dual conduction pathways, perfect proton selectivity, a single‐channel conductance approximately 103 times smaller than most ion channels, voltage‐dependent gating that is strongly modulated by the pH gradient, ΔpH, and potent inhibition by Zn2+ (in many species) but an absence of other potent inhibitors. The recent identification of HV1 in three unicellular marine plankton species has dramatically expanded the phylogenetic family tree. Interest in proton channels in their own right has increased as important physiological roles have been identified in many cells. Proton channels trigger the bioluminescent flash of dinoflagellates, facilitate calcification by coccolithophores, regulate pH‐dependent processes in eggs and sperm during fertilization, secrete acid to control the pH of airway fluids, facilitate histamine secretion by basophils, and play a signaling role in facilitating B‐cell receptor mediated responses in B‐lymphocytes. The most elaborate and best‐established functions occur in phagocytes, where proton channels optimize the activity of NADPH oxidase, an important producer of reactive oxygen species. Proton efflux mediated by HV1 balances the charge translocated across the membrane by electrons through NADPH oxidase, minimizes changes in cytoplasmic and phagosomal pH, limits osmotic swelling of the phagosome, and provides substrate H+ for the production of H2O2 and HOCl, reactive oxygen species crucial to killing pathogens. © 2012 American Physiological Society. Compr Physiol 2:1355‐1385, 2012.

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Figure 1. Figure 1.

Voltage‐gated proton channels trigger the bioluminescent light flash in dinoflagellates. The proton concentration is high in the flotation vacuole (below the membrane in the diagram). An action potential depolarizes the membrane, opening proton channels, allowing protons to flow down their electrochemical gradient into the interior of the scintillon (upper compartment). The sudden drop in pH in the scintillon triggers the flash by two concerted mechanisms. Upon protonation, the light‐emitting pigment, luciferin (LH2) is released from luciferin‐binding protein (LBP) making it available as a substrate for luciferase, which is itself activated by acidification. [Adapted, with permission, from reference 109. Originally published in Bioluminescence in Action, edited by P. Herring, Academic Press, London. pp. 129‐170, 1978. Copyright Elsevier.]
Figure 2. Figure 2.

Membrane topology of voltage‐gated K+ channels (left), voltage‐gated proton channels (center), and voltage‐sensitive phosphatases (right). The K+ channel (lower panel) is a tetramer of the six transmembrane domain monomer shown in the top; the proton channel is a homodimer of the four transmembrane domain monomer in the top panel, and the phosphatase is a monomer. The assembled K+ channel has a single conduction pathway, but the proton channel has two, one in each protomer. The voltage‐sensing phosphatase (VSP) senses voltage, but does not conduct. The proton channel cartoon illustrates schematically that the dimer is held together by C‐terminal coiled‐coil interactions, and that the channel can be phosphorylated at Thr29 in the intracellular N terminus to produce the enhanced gating mode (cf. Figure 20). [Figure adapted, with permission, from reference 58. Originally published in Physiology 25:27‐40, 2010.]
Figure 3. Figure 3.

A phylogenetic analysis showing evolutionary relationships among 37 putative HV1 voltage‐sensing domain (VSD) sequences. This maximum likelihood phylogenetic tree with 100 boostraps was constructed from a multiple sequence alignment of the VSD portion of 37 HV1s; N‐ and C‐termini were truncated. Branch lengths are displayed to scale, and are proportional to the evolutionary distance between sequences. Bootstrap values greater than 60 are shown. [Figure adapted, with permission, from reference 251. Originally published in Proc. Natl. Acad. Sci. U S A. 108:18162‐18168, 2011.].
Figure 4. Figure 4.

Comparison of gating kinetics of the human proton channel dimer (A) and monomer (B), expressed in HEK‐293 cells. Note the slower, sigmoid activation of the WT channel (A), and the faster, exponential turn‐on of the monomer (C terminus truncated, HV1ΔC) both during pulses to +50 mV at 23°C at pHo 7.5, pHi 7.5. [Adapted from reference 199. Originally published in The Journal of Physiology.] (C) The time course of movement of a fluorescent probe attached to the S4 domain of the Ciona proton channel is exponential (red). This time course raised to the second power (green) matches the proton current recorded at the same time (black). [Adapted, with permission, from reference 103. Adapted, with permission, from Macmillan Publishers Ltd: Nature Structural & Molecular Biology 17: 51‐56, 2010.]
Figure 5. Figure 5.

Possible dimer interfaces, with the transmembrane domains color coded as: S1 red, S2 yellow, S3 green, and S4 blue. The dimer in A was proposed on the basis of cysteine cross‐linking studies 150; the dimer in B was proposed to explain Zn2+ binding studies 199. External His residues that bind Zn2+ 224 are shown in aqua (cf. Figure 14. [Adapted, with permission, from reference 198. Originally published in Channels (Austin) 4: 260‐265, 2010.]
Figure 6. Figure 6.

Hydrogen‐bonded chain (HBC) conduction. In this schematic example, hydroxyl groups (e.g., from Ser residues) form a HBC that spans the membrane. (A) Proton conduction occurs when a proton enters the chain from the left to form a positive ion, which then moves to the right by hopping of successive protons to effect a reversal of the direction of the hydrogen bonds between each pair of oxygen atoms. Proton conduction would also occur by loss of a proton on the right followed by movement of a negative ion (or fault) to the left. (B) To complete the process, reorientation of each hydrogen bond in the chain must occur, so that another proton can enter from the same side. [Redrawn, with permission, from reference 202.]
Figure 7. Figure 7.

Determination of the single channel conductance from proton current fluctuations that reflect the stochastic opening and closing of proton channels. “A” shows families of proton currents in an excised, inside‐out patch from a human eosinophil at three pHi values. At subthreshold voltages, the current is quiet, but just above Vthreshold, proton channels begin to open, and the current becomes distinctly noisy. It is noteworthy that (at low pHi) the noise first increases with depolarization, but then decreases for large depolarizations. “B” shows gH‐V relationships from this patch. The variance of the current fluctuation, measured at quasi‐steady‐state, is plotted in “C.” The variance increases more than 100‐fold at voltages where the proton conductance is active, and is maximal near the midpoint of the gH‐V relationship at each pHi (indicated as V1/2). “D” shows that the expected variance given the simplest possible two‐state model of gating (closed ↔ open) coincides with the observed voltage dependence. The nonmonotonic increase in variance with depolarization is consistent with the maximum Popen limiting to approximately 0.95 at pHi 5.5.

Adapted, with permission, from reference 39 © Cherny et al., 2003. Originally published in The Journal of General Physiology.
Figure 8. Figure 8.

(A‐D) Effects of pHi on voltage‐gated proton currents in an inside‐out membrane patch from a rat alveolar epithelial cell. The pipette pH (i.e., pHo) was 7.5. “A” shows proton currents in a cell‐attached patch that increase gradually with time during each pulse because the single channel proton currents are too small to resolve. Superimposed are large single channel currents most likely due to Kv1.3 delayed rectifier channels that dominate macroscopic currents in these cells 71. After this patch was excised into K+‐free solutions, the same population of proton channels generated the currents shown in “B‐D” at the indicated pHi. As pHi was decreased, the currents became larger and activated much more rapidly (note the changing calibrations). The graph in E shows average activation time constants (τact) obtained by single exponential fits in many patches. Changing pHo shifts the kinetics along the voltage axis with little change in the range of τact values. In contrast, changes in pHi profoundly affect τact, with an approximately 5‐fold slowing per unit increase in pHi.

Adapted, with permission, from reference 63. Originally published in The Journal of Physiology 489:299‐307, 1995.
Figure 9. Figure 9.

(A) Families of proton currents at different pHo//pHi in three rat alveolar epithelial cells (one in each row). The most obvious effect of pH is to vary the voltage range in which proton channels open. (B) Average current‐voltage relationships at the indicated ΔpH, where ΔpH = pHo − pHi, illustrate that the position of the gH‐V relationship is completely determined by ΔpH. The “Rule of Forty” (Eqs. 1,2,3) expresses the 40‐mV shift in the position of the gH‐V relationship that occurs when ΔpH changes by one unit, regardless of whether pHo or pHi is changed.

Adapted, with permission, from reference 38 © Cherny et al., 1995. Originally published in The Journal of General Physiology 105:861‐896.
Figure 10. Figure 10.

Regulation of the position of the gH‐V relationship by ΔpH occurs in all known voltage‐gated proton channels. In most species, this regulation results in Vthreshold always being positive to the reversal potential, Vrev, thus ensuring that when proton channels open, acid will be extruded. The blue line shows the result of linear regression on data reported in 15 cell types that are listed in reference 56; the dashed red line shows equality between Vthreshold and Vrev for comparison. In the recently identified kHv1 channel in the dinoflagellate, Karlodiniun veneficum, Vthreshold is shifted by −60 mV relative to all other species (green data points and line), with the result that depolarization above Vthreshold will produce inward H+ current in kHV1 at all ΔpH 251.
Figure 11. Figure 11.

Proton channel gating is strongly temperature dependent. (A, B) In a human neutrophil at pHo 7.0, pHi 5.5 at the indicated temperatures, a depolarizing prepulse activated the gH, then the voltage was stepped to test voltages, illustrated in 20‐mV increments. Note the change in time base. The “tail current” (deactivation or closing) time constant, τtail, was obtained by fitting a single decaying exponential to each current. The Q10 was identical at all voltages (C) and was 8.5 in this cell, corresponding to an activation energy of approximately 38 kcal/mol.

Adapted, with permission, from reference 67 © DeCoursey & Cherny, 1998. Originally published in The Journal of General Physiology.
Figure 12. Figure 12.

Modulation of Zn2+ effects on proton currents by pHo reveals strong competition between Zn2+ and H+ for external binding sites. Identical families of pulses were applied in each row at the indicated pHo and Zn2+ concentrations. Zn2+ slows activation of the proton current at a given voltage and shifts the gH‐V relationship positively.

Adapted, with permission, from reference 36 © Cherny & DeCoursey, 1999. Originally published in The Journal of General Physiology.
Figure 13. Figure 13.

The pHo dependence of the slowing of proton current activation can be explained if Zn2+ binds to a site consisting of three groups with pKa 6.3, with affinity 316 nmol/L, and cooperativity factor a = 0.03 (see text). All curves were drawn with these values and no other adjustable parameters. Adequate fits were also obtained by assuming two titratable groups, but not with only one. At high pHo competition with protons disappears and limits to simple metal binding. See text for further details.

Adapted, with permission, from reference 36 © Cherny & DeCoursey, 1999. Originally published in The Journal of General Physiology.
Figure 14. Figure 14.

Effects of Zn2+ on Vthreshold (A) and on the slowing of proton current activation (B) in constructs with several His mutations. Dimeric proton channels are illustrated with diagrams in which solid symbols indicate His and open symbols indicate Ala substituted for His. Three tandem dimers are shown with their C‐ and N‐termini linked to constrain the His positions in the dimer. Vthreshold shifts were estimated from gH‐V relationships plotted semilogarithmically. Statistical comparisons are to WT channel parameters (P < 0.05, *P < 0.01).

Adapted, with permission, from reference 199. Originally published in The Journal of Physiology 588:1435‐1449, 2010.
Figure 15. Figure 15.

Proposed mechanism by which proton flux through voltage‐gated proton channels triggers light flashes in dinoflagellates. Mechanical stimulation elicits an action potential conducted along the tonoplast, the membrane separating the central vacuole and a thin layer of cytoplasm (gray). When the action potential invades the scintillons (grape‐like structures formed by evagination of tonoplast), proton flux from the vacuole at low pH into the scintillon activates luciferase, triggering the flash.

Adapted, with permission, from reference 110. Originally published in Hastings JW. Bioluminescence. In: Cell Physiology Sourcebook: A Molecular Approach (3rd ed.), Sperelakis N, editor. San Diego: Academic Press, 2001, p. 1115‐1131.
Figure 16. Figure 16.

Rapidly activating proton currents in a Lymnaea snail neuron, 120 μm in diameter. Currents during pulses to the voltages shown at pHo 7.4, pHi 5.9 at room temperature.

Originally published in The Journal of Physiology

351:199‐216, 1984.]

Adapted, with permission, from reference 29.
Figure 17. Figure 17.

Schematic representation of the role of the proton channel (HVCN1) in the context of B cell receptor (BCR) stimulation. Antigen binding to the BCR results in phosphorylation of immunoreceptor tyrosine activation motif (ITAMs) in the Ig‐α/β heterodimer by LYN, creating docking sites for Syk. This serves to amplify BCR signaling by further recruitment and activation of Syk, which leads to PI3K activation, activation of Akt and increased glucose uptake and metabolism. Amplification of signaling is negatively regulated by CD22, which is also phosphorylated by LYN, providing a docking site for protein tyrosine phosphatase SHP‐1. In resting cells, SHP‐1 inhibits B cell activation by dephosphorylating Syk, thus counterbalancing ITAM‐Syk mediated signal amplification. BCR stimulation results in reactive oxygen species (ROS) generated by NADPH oxidase. The O2.− that is produced combines with protons to form H2O2 and O2, which freely diffuse through the membrane. ROS generate a localized oxidizing environment causing inhibition of SHP‐1, which results in amplification of BCR signal. HVCN1 sustains NADPH oxidase activity. In the absence of HVCN1, the oxidizing environment cannot be maintained and consequently SHP‐1 remains active, reducing BCR‐signal strength.

Adapted, with permission, from reference 32. Originally published in Nature Immunology 11: 265‐272, 2010.]
Figure 18. Figure 18.

Proton husbandry during the phagocyte respiratory burst. Upon stimulation, the components of the NADPH oxidase complex assemble at the membrane and begin to relay electrons from NADPH across the membrane to reduce O2 to superoxide anion, O2.−. Because of the massive increase in O2 consumption, this process is called the “respiratory burst” 12 despite the fact that most of the O2 is consumed to make reactive oxygen species (ROS) and it is not affected by mitochondrial inhibitors 235. Protons left behind in the cytoplasm exit mainly through voltage‐gated proton channels 193, although other transporters also play important roles 178. Inside the phagosome, protons are consumed during the spontaneous dismutation of O2.− to H2O2 as well as during HOCl generation by myeloperoxidase (MPO) 138. [Adapted, with permission, from reference 58. Originally published in Physiology 25: 27‐40, 2010.]
Figure 19. Figure 19.

The pHi in four individual human neutrophils during phagocytosis of opsonized zymosan (OPZ) is plotted, with pseudocolor confocal images of each cell at corresponding times. The cells were loaded with the pH sensing dye, SNARF‐1. In control cells, pHi drops rapidly when OPZ is ingested and often recovers rapidly (A), but in some cells does not (B). Recovery is prevented by 20 μmol/L dimethylamiloride, which inhibits the Na+/H+ antiporter (C) or 100 μmol/L Zn2+, which inhibits proton channels (D). Among the inhibitors tested, only Zn2+ increased the rate of acidification, indicating that proton channels contribute significantly at early times. [Adapted, with permission, from reference 178. Originally published in Proceedings of the National Academy of Sciences, USA 106: 18022‐18027, 2009.]
Figure 20. Figure 20.

The enhanced gating mode of proton channels in human eosinophils. (A) The onset of enhanced gating in a human eosinophil stimulated with 30 nmol/L PMA in perforated‐patch configuration at symmetrical pH 7.0. Test pulses to +60 mV applied at 30‐s intervals are superimposed, before and up to 6 min after addition of PMA, illustrating the increasing current, faster activation, and slower tail current decay. The downward shift of the baseline current at −60 mV reflects electron current generated by NADPH oxidase activity. (B) The time course of proton current enhancement (top) after stimulation with PMA (arrow) and subsequent inhibition of PKC by 3 μmol/L GF109203X (GFX) (arrow) in a different human eosinophil. Repeated test pulses to +60 mV were applied, and only the peak current is evident at this time scale. The inward electron current at −60 mV in this cell can be seen as a downward deflection in the holding current, which is amplified in the lower panel (the pulses eliciting proton current are blanked). “C” shows individual currents during test pulses applied at times indicated in “B” by lower‐case letters. The inward electron current at the holding potential is clearly evident. [Adapted, with permission, from reference 68. (A) Originally published in The Journal of Physiology 535: 767‐781, 2001 and from reference 179 (B, C) Originally published in The Journal of Physiology 579: 327‐344, 2007.]
Figure 21. Figure 21.

Confocal images of four human basophils taken at 30‐s intervals (left to right, then top to bottom) during their response to anti‐IgE stimulation. The pseudocolors indicate pHi detected with SNARF‐1 in confocal slices approximately 0.2 μm thick, with alkaline to acidic shown as blue to green to red to yellow. Voltage‐gated proton channels are active during the response and limit the extent of acidification. (Image provided by Deri Morgan with permission.)


Figure 1.

Voltage‐gated proton channels trigger the bioluminescent light flash in dinoflagellates. The proton concentration is high in the flotation vacuole (below the membrane in the diagram). An action potential depolarizes the membrane, opening proton channels, allowing protons to flow down their electrochemical gradient into the interior of the scintillon (upper compartment). The sudden drop in pH in the scintillon triggers the flash by two concerted mechanisms. Upon protonation, the light‐emitting pigment, luciferin (LH2) is released from luciferin‐binding protein (LBP) making it available as a substrate for luciferase, which is itself activated by acidification. [Adapted, with permission, from reference 109. Originally published in Bioluminescence in Action, edited by P. Herring, Academic Press, London. pp. 129‐170, 1978. Copyright Elsevier.]



Figure 2.

Membrane topology of voltage‐gated K+ channels (left), voltage‐gated proton channels (center), and voltage‐sensitive phosphatases (right). The K+ channel (lower panel) is a tetramer of the six transmembrane domain monomer shown in the top; the proton channel is a homodimer of the four transmembrane domain monomer in the top panel, and the phosphatase is a monomer. The assembled K+ channel has a single conduction pathway, but the proton channel has two, one in each protomer. The voltage‐sensing phosphatase (VSP) senses voltage, but does not conduct. The proton channel cartoon illustrates schematically that the dimer is held together by C‐terminal coiled‐coil interactions, and that the channel can be phosphorylated at Thr29 in the intracellular N terminus to produce the enhanced gating mode (cf. Figure 20). [Figure adapted, with permission, from reference 58. Originally published in Physiology 25:27‐40, 2010.]



Figure 3.

A phylogenetic analysis showing evolutionary relationships among 37 putative HV1 voltage‐sensing domain (VSD) sequences. This maximum likelihood phylogenetic tree with 100 boostraps was constructed from a multiple sequence alignment of the VSD portion of 37 HV1s; N‐ and C‐termini were truncated. Branch lengths are displayed to scale, and are proportional to the evolutionary distance between sequences. Bootstrap values greater than 60 are shown. [Figure adapted, with permission, from reference 251. Originally published in Proc. Natl. Acad. Sci. U S A. 108:18162‐18168, 2011.].



Figure 4.

Comparison of gating kinetics of the human proton channel dimer (A) and monomer (B), expressed in HEK‐293 cells. Note the slower, sigmoid activation of the WT channel (A), and the faster, exponential turn‐on of the monomer (C terminus truncated, HV1ΔC) both during pulses to +50 mV at 23°C at pHo 7.5, pHi 7.5. [Adapted from reference 199. Originally published in The Journal of Physiology.] (C) The time course of movement of a fluorescent probe attached to the S4 domain of the Ciona proton channel is exponential (red). This time course raised to the second power (green) matches the proton current recorded at the same time (black). [Adapted, with permission, from reference 103. Adapted, with permission, from Macmillan Publishers Ltd: Nature Structural & Molecular Biology 17: 51‐56, 2010.]



Figure 5.

Possible dimer interfaces, with the transmembrane domains color coded as: S1 red, S2 yellow, S3 green, and S4 blue. The dimer in A was proposed on the basis of cysteine cross‐linking studies 150; the dimer in B was proposed to explain Zn2+ binding studies 199. External His residues that bind Zn2+ 224 are shown in aqua (cf. Figure 14. [Adapted, with permission, from reference 198. Originally published in Channels (Austin) 4: 260‐265, 2010.]



Figure 6.

Hydrogen‐bonded chain (HBC) conduction. In this schematic example, hydroxyl groups (e.g., from Ser residues) form a HBC that spans the membrane. (A) Proton conduction occurs when a proton enters the chain from the left to form a positive ion, which then moves to the right by hopping of successive protons to effect a reversal of the direction of the hydrogen bonds between each pair of oxygen atoms. Proton conduction would also occur by loss of a proton on the right followed by movement of a negative ion (or fault) to the left. (B) To complete the process, reorientation of each hydrogen bond in the chain must occur, so that another proton can enter from the same side. [Redrawn, with permission, from reference 202.]



Figure 7.

Determination of the single channel conductance from proton current fluctuations that reflect the stochastic opening and closing of proton channels. “A” shows families of proton currents in an excised, inside‐out patch from a human eosinophil at three pHi values. At subthreshold voltages, the current is quiet, but just above Vthreshold, proton channels begin to open, and the current becomes distinctly noisy. It is noteworthy that (at low pHi) the noise first increases with depolarization, but then decreases for large depolarizations. “B” shows gH‐V relationships from this patch. The variance of the current fluctuation, measured at quasi‐steady‐state, is plotted in “C.” The variance increases more than 100‐fold at voltages where the proton conductance is active, and is maximal near the midpoint of the gH‐V relationship at each pHi (indicated as V1/2). “D” shows that the expected variance given the simplest possible two‐state model of gating (closed ↔ open) coincides with the observed voltage dependence. The nonmonotonic increase in variance with depolarization is consistent with the maximum Popen limiting to approximately 0.95 at pHi 5.5.

Adapted, with permission, from reference 39 © Cherny et al., 2003. Originally published in The Journal of General Physiology.


Figure 8.

(A‐D) Effects of pHi on voltage‐gated proton currents in an inside‐out membrane patch from a rat alveolar epithelial cell. The pipette pH (i.e., pHo) was 7.5. “A” shows proton currents in a cell‐attached patch that increase gradually with time during each pulse because the single channel proton currents are too small to resolve. Superimposed are large single channel currents most likely due to Kv1.3 delayed rectifier channels that dominate macroscopic currents in these cells 71. After this patch was excised into K+‐free solutions, the same population of proton channels generated the currents shown in “B‐D” at the indicated pHi. As pHi was decreased, the currents became larger and activated much more rapidly (note the changing calibrations). The graph in E shows average activation time constants (τact) obtained by single exponential fits in many patches. Changing pHo shifts the kinetics along the voltage axis with little change in the range of τact values. In contrast, changes in pHi profoundly affect τact, with an approximately 5‐fold slowing per unit increase in pHi.

Adapted, with permission, from reference 63. Originally published in The Journal of Physiology 489:299‐307, 1995.


Figure 9.

(A) Families of proton currents at different pHo//pHi in three rat alveolar epithelial cells (one in each row). The most obvious effect of pH is to vary the voltage range in which proton channels open. (B) Average current‐voltage relationships at the indicated ΔpH, where ΔpH = pHo − pHi, illustrate that the position of the gH‐V relationship is completely determined by ΔpH. The “Rule of Forty” (Eqs. 1,2,3) expresses the 40‐mV shift in the position of the gH‐V relationship that occurs when ΔpH changes by one unit, regardless of whether pHo or pHi is changed.

Adapted, with permission, from reference 38 © Cherny et al., 1995. Originally published in The Journal of General Physiology 105:861‐896.


Figure 10.

Regulation of the position of the gH‐V relationship by ΔpH occurs in all known voltage‐gated proton channels. In most species, this regulation results in Vthreshold always being positive to the reversal potential, Vrev, thus ensuring that when proton channels open, acid will be extruded. The blue line shows the result of linear regression on data reported in 15 cell types that are listed in reference 56; the dashed red line shows equality between Vthreshold and Vrev for comparison. In the recently identified kHv1 channel in the dinoflagellate, Karlodiniun veneficum, Vthreshold is shifted by −60 mV relative to all other species (green data points and line), with the result that depolarization above Vthreshold will produce inward H+ current in kHV1 at all ΔpH 251.



Figure 11.

Proton channel gating is strongly temperature dependent. (A, B) In a human neutrophil at pHo 7.0, pHi 5.5 at the indicated temperatures, a depolarizing prepulse activated the gH, then the voltage was stepped to test voltages, illustrated in 20‐mV increments. Note the change in time base. The “tail current” (deactivation or closing) time constant, τtail, was obtained by fitting a single decaying exponential to each current. The Q10 was identical at all voltages (C) and was 8.5 in this cell, corresponding to an activation energy of approximately 38 kcal/mol.

Adapted, with permission, from reference 67 © DeCoursey & Cherny, 1998. Originally published in The Journal of General Physiology.


Figure 12.

Modulation of Zn2+ effects on proton currents by pHo reveals strong competition between Zn2+ and H+ for external binding sites. Identical families of pulses were applied in each row at the indicated pHo and Zn2+ concentrations. Zn2+ slows activation of the proton current at a given voltage and shifts the gH‐V relationship positively.

Adapted, with permission, from reference 36 © Cherny & DeCoursey, 1999. Originally published in The Journal of General Physiology.


Figure 13.

The pHo dependence of the slowing of proton current activation can be explained if Zn2+ binds to a site consisting of three groups with pKa 6.3, with affinity 316 nmol/L, and cooperativity factor a = 0.03 (see text). All curves were drawn with these values and no other adjustable parameters. Adequate fits were also obtained by assuming two titratable groups, but not with only one. At high pHo competition with protons disappears and limits to simple metal binding. See text for further details.

Adapted, with permission, from reference 36 © Cherny & DeCoursey, 1999. Originally published in The Journal of General Physiology.


Figure 14.

Effects of Zn2+ on Vthreshold (A) and on the slowing of proton current activation (B) in constructs with several His mutations. Dimeric proton channels are illustrated with diagrams in which solid symbols indicate His and open symbols indicate Ala substituted for His. Three tandem dimers are shown with their C‐ and N‐termini linked to constrain the His positions in the dimer. Vthreshold shifts were estimated from gH‐V relationships plotted semilogarithmically. Statistical comparisons are to WT channel parameters (P < 0.05, *P < 0.01).

Adapted, with permission, from reference 199. Originally published in The Journal of Physiology 588:1435‐1449, 2010.


Figure 15.

Proposed mechanism by which proton flux through voltage‐gated proton channels triggers light flashes in dinoflagellates. Mechanical stimulation elicits an action potential conducted along the tonoplast, the membrane separating the central vacuole and a thin layer of cytoplasm (gray). When the action potential invades the scintillons (grape‐like structures formed by evagination of tonoplast), proton flux from the vacuole at low pH into the scintillon activates luciferase, triggering the flash.

Adapted, with permission, from reference 110. Originally published in Hastings JW. Bioluminescence. In: Cell Physiology Sourcebook: A Molecular Approach (3rd ed.), Sperelakis N, editor. San Diego: Academic Press, 2001, p. 1115‐1131.


Figure 16.

Rapidly activating proton currents in a Lymnaea snail neuron, 120 μm in diameter. Currents during pulses to the voltages shown at pHo 7.4, pHi 5.9 at room temperature.

Originally published in The Journal of Physiology

351:199‐216, 1984.]

Adapted, with permission, from reference 29.


Figure 17.

Schematic representation of the role of the proton channel (HVCN1) in the context of B cell receptor (BCR) stimulation. Antigen binding to the BCR results in phosphorylation of immunoreceptor tyrosine activation motif (ITAMs) in the Ig‐α/β heterodimer by LYN, creating docking sites for Syk. This serves to amplify BCR signaling by further recruitment and activation of Syk, which leads to PI3K activation, activation of Akt and increased glucose uptake and metabolism. Amplification of signaling is negatively regulated by CD22, which is also phosphorylated by LYN, providing a docking site for protein tyrosine phosphatase SHP‐1. In resting cells, SHP‐1 inhibits B cell activation by dephosphorylating Syk, thus counterbalancing ITAM‐Syk mediated signal amplification. BCR stimulation results in reactive oxygen species (ROS) generated by NADPH oxidase. The O2.− that is produced combines with protons to form H2O2 and O2, which freely diffuse through the membrane. ROS generate a localized oxidizing environment causing inhibition of SHP‐1, which results in amplification of BCR signal. HVCN1 sustains NADPH oxidase activity. In the absence of HVCN1, the oxidizing environment cannot be maintained and consequently SHP‐1 remains active, reducing BCR‐signal strength.

Adapted, with permission, from reference 32. Originally published in Nature Immunology 11: 265‐272, 2010.]


Figure 18.

Proton husbandry during the phagocyte respiratory burst. Upon stimulation, the components of the NADPH oxidase complex assemble at the membrane and begin to relay electrons from NADPH across the membrane to reduce O2 to superoxide anion, O2.−. Because of the massive increase in O2 consumption, this process is called the “respiratory burst” 12 despite the fact that most of the O2 is consumed to make reactive oxygen species (ROS) and it is not affected by mitochondrial inhibitors 235. Protons left behind in the cytoplasm exit mainly through voltage‐gated proton channels 193, although other transporters also play important roles 178. Inside the phagosome, protons are consumed during the spontaneous dismutation of O2.− to H2O2 as well as during HOCl generation by myeloperoxidase (MPO) 138. [Adapted, with permission, from reference 58. Originally published in Physiology 25: 27‐40, 2010.]



Figure 19.

The pHi in four individual human neutrophils during phagocytosis of opsonized zymosan (OPZ) is plotted, with pseudocolor confocal images of each cell at corresponding times. The cells were loaded with the pH sensing dye, SNARF‐1. In control cells, pHi drops rapidly when OPZ is ingested and often recovers rapidly (A), but in some cells does not (B). Recovery is prevented by 20 μmol/L dimethylamiloride, which inhibits the Na+/H+ antiporter (C) or 100 μmol/L Zn2+, which inhibits proton channels (D). Among the inhibitors tested, only Zn2+ increased the rate of acidification, indicating that proton channels contribute significantly at early times. [Adapted, with permission, from reference 178. Originally published in Proceedings of the National Academy of Sciences, USA 106: 18022‐18027, 2009.]



Figure 20.

The enhanced gating mode of proton channels in human eosinophils. (A) The onset of enhanced gating in a human eosinophil stimulated with 30 nmol/L PMA in perforated‐patch configuration at symmetrical pH 7.0. Test pulses to +60 mV applied at 30‐s intervals are superimposed, before and up to 6 min after addition of PMA, illustrating the increasing current, faster activation, and slower tail current decay. The downward shift of the baseline current at −60 mV reflects electron current generated by NADPH oxidase activity. (B) The time course of proton current enhancement (top) after stimulation with PMA (arrow) and subsequent inhibition of PKC by 3 μmol/L GF109203X (GFX) (arrow) in a different human eosinophil. Repeated test pulses to +60 mV were applied, and only the peak current is evident at this time scale. The inward electron current at −60 mV in this cell can be seen as a downward deflection in the holding current, which is amplified in the lower panel (the pulses eliciting proton current are blanked). “C” shows individual currents during test pulses applied at times indicated in “B” by lower‐case letters. The inward electron current at the holding potential is clearly evident. [Adapted, with permission, from reference 68. (A) Originally published in The Journal of Physiology 535: 767‐781, 2001 and from reference 179 (B, C) Originally published in The Journal of Physiology 579: 327‐344, 2007.]



Figure 21.

Confocal images of four human basophils taken at 30‐s intervals (left to right, then top to bottom) during their response to anti‐IgE stimulation. The pseudocolors indicate pHi detected with SNARF‐1 in confocal slices approximately 0.2 μm thick, with alkaline to acidic shown as blue to green to red to yellow. Voltage‐gated proton channels are active during the response and limit the extent of acidification. (Image provided by Deri Morgan with permission.)

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Further Reading
 1. Bezanilla F. The voltage sensor in voltage‐dependent ion channels. Physiol Rev 80: 555‐592, 2000.
 2. Börjesson, SI, Elinder F. Structure, function, and modification of the voltage sensor in voltage‐gated ion channels. Cell Biochem Biophys 52: 149‐174, 2008.
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 5. Capasso M, DeCoursey TE, Dyer MJS. pH regulation and beyond: Unanticipated functions for the voltage‐gated proton channel, HVCN1. Trends Cell Biol. 21: 20‐28, 2011.
 6. DeCoursey TE. Voltage‐gated proton channels and other proton transfer pathways. Physiol Rev 83: 475‐579, 2003.
 7.Demaurex N, El Chemaly A. Physiological roles of voltage‐gated proton channels in leukocytes. J Physiol 588: 4659‐4665, 2010.
 8.Kirichok, Y, Lishko PV. Rediscovering sperm ion channels with the patch‐clamp technique. Mol Hum Reprod 17: 478‐499, 2011.
 9. Fischer, H. Function of proton channels in lung epithelia. WIRES Membr Transp Signal (in press), 2011. doi: 10.1002/wmts.17
 10. Hille B. 2001. Ion Channels of Excitable Membranes. (3rd ed). Sunderland, MA: Sinauer Associates, Inc. p. 814.
 11.Okamura Y, Murata Y, Iwasaki H. Voltage‐sensing phosphatase: Actions and potentials. J Physiol 587: 513‐520, 2009.
 12.Okamura Y. Biodiversity of voltage sensor domain proteins. Pflügers Arch 454: 361‐371, 2007.

Further Reading 

Reviews of voltage sensing mechanisms in ion channels:

Bezanilla F. The voltage sensor in voltage-dependent ion channels. Physiol Rev 80: 555-592, 2000.

Börjesson, S.I., and F. Elinder. 2008. Structure, function, and modification of the voltage sensor in voltage-gated ion channels. Cell. Biochem. Biophys. 52:149-174.

Swartz KJ. Sensing voltage across lipid membranes. Nature 456: 891-897, 2008.

Tombola F, Pathak MM, and Isacoff EY. How does voltage open an ion channel? Annu Rev Cell Dev Biol 22: 23-52, 2006.

A recent update on proposed functions of proton channels in various cells:

Capasso M, DeCoursey TE, Dyer MJS.  pH regulation and beyond: unanticipated functions for the voltage-gated proton channel, HVCN1.  Trends Cell Biol.  21:20-28, 2011.  doi:10.1016/j.tcb.2010.09.006

This exhaustive review of voltage-gated proton channels includes comparisons with several other proton conducting molecules:

DeCoursey TE. Voltage-gated proton channels and other proton transfer pathways. Physiol Rev 83: 475-579, 2003.

A recent review of functions of proton channels in phagocytes:

Demaurex N, and El Chemaly A. Physiological roles of voltage-gated proton channels in leukocytes. J Physiol 588:4659-4665, 2010.  doi: 10.1113/jphysiol.2010.194225

A recent focused review of functions of proton channels in sperm:

Kirichok, Y., and P.V. Lishko. 2011. Rediscovering sperm ion channels with the patch-clamp technique. Mol. Hum. Reprod. 17:478-499.

A recent review of functions of proton channels in epithelium:

Fischer, H. 2011. Functions of proton channels in epithelial cells. WIRES Membrane Transport and Signaling. In press.

This is the classic textbook on ion channels:

Hille B. 2001. Ion Channels of Excitable Membranes. 3rd ed. Sinauer Associates, Inc. Sunderland, MA. 814 pp.

Reviews of voltage-sensing phosphatases:

Okamura Y, Murata Y, Iwasaki H. Voltage-sensing phosphatase: actions and potentials. J Physiol 587: 513-520, 2009.

Okamura Y. Biodiversity of voltage sensor domain proteins. Pflügers Arch 454: 361-371, 2007.

 


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Article Title: Voltage‐Gated Proton Channels
 

How to Cite

Thomas E. DeCoursey. Voltage‐Gated Proton Channels. Compr Physiol 2012, 2: 1355-1385. doi: 10.1002/cphy.c100071