Proton channels in non-phagocytic cells of the immune system

Abstract

Proton channels are expressed in all cells of the immune system to various degrees. While their function in phagocytic cells, immune cells that engulf bacteria and cell debris for clearance, has been the object of extensive research, the function of proton channels in non-phagocytic cells has remained more elusive until recently. Further studies have been helped by the discovery of the gene coding for the mammalian proton channel, HVCN1, which has prompted a new wave of research in this area. Recent findings show how proton channels regulate cell function in non-phagocytic cells of the immune system such as basophils and lymphocytes.

The gene coding for the voltage-gated proton channel, HVCN1, was described for the first time in 2006, almost 20 years after the existence of proton currents in human cells was first proposed (1, 2). The highly proton-selective channel, also called H_v1 for the human protein (3) and mVSOP for the mouse homologue (4), is a four-transmembrane domain protein, similar to the voltage-sensor domain (VSD) of voltage-gated cation channels (Fig. 1). Unlike most voltage gated ion channels, HVCN1 does not have separate voltage-sensing and pore-forming domains; the conduction pathway is contained within the VSD. The ion selectivity is determined by amino acid residues in transmembrane domains, Asp¹¹² in the first transmembrane domain of the human channel, in particular (5). Mutation of this residue results in abrogation of proton-selective currents, indicating the side-chain of Asp¹¹² plays a fundamental role in determining the channel proton conductance. Intriguingly, this mutation not only abrogates proton conductance but also renders HVCN1 an anion-selective channel (5). Other amino acid residues have been described to play a role in channel regulation. Two His residues, His¹⁴⁰ and His¹⁹³, predicted to reside within or in close proximity to the two extracellular loops of the protein, bind divalent cations, such as Zn²⁺ (3), shown to be strong inhibitors of proton currents. Studies of an homology structure of HVCN1 transmembrane domains, derived from the voltage-sensing domain of voltage-gated potassium channels, revealed that the distance between the two His residues is too long to accommodate a Zn²⁺ ion, suggesting that the ion binds to His residues on separate molecules (6), since HVCN1 exists as a dimer (7–9).

Figure 1. Amino acid sequence of human HVCN1.

The threonine residue in the intracellular N-terminus domain (Thr²⁹, highlighted) is important for channel function, since its phosphorylation enhances channel opening in leukocytes (23). Asp¹¹², on the other hand, is responsible for proton selectivity (5). The two histidines constituting Zn²⁺ binding site are indicated (3), together with transmembrane domains (four rectangular boxes). Figure adapted from Trends in Cell Biology (43).

From a functional perspective, proton currents have been studied mostly in phagocytic cells (10). However, other cells of the immune system express proton channels and while their function in some of them has been characterized recently, such as basophils (11) and B lymphocytes (12), their role in other cell types such as T lymphocytes remains more elusive. This review will highlight the significance of proton channels in non-phagocytic cells of the immune system and discuss possible roles not yet completely elucidated.

HVCN1 in basophils

Basophils, which normally comprise less than 1% of circulating leukocytes, differentiate from the same common myeloid precursor as neutrophils and eosinophils. Like these other myeloid cells, they contain numerous mediator-rich cytoplasmic granules, thus leading to the common description of neutrophils, eosinophils, and basophils as granulocytes. One of several distinctions between basophils and either neutrophils and eosinophils, however, is that basophils do not express the enzyme NADPH oxidase (13). This enzymatic complex assembles on the plasma or phagosome membrane of phagocytic cells when they engulf bacteria and is responsible for the production of superoxide anion, O₂^•−, a precursor to other reactive oxygen species (ROS). ROS are oxidizing agents and their production in phagocytic cells is required for microbial killing, as exemplified by the impaired immune responses observed in chronic granulomatous disease (CGD) patients, whose immune cells lack a functional NADPH oxidase (14). The impairment in CGD lies mainly with the phagocytic cells, although B cell responses are also altered in these patients (15). As will be discussed later, NADPH oxidase-dependent ROS production is important not only in phagocytic cells to clear bacteria but also in B cells to sustain B cell activation (12). The activity of the NADPH oxidase is electrogenic, transferring negative charges (electrons) taken from cytoplasmic NADPH to extracellular or phagosomal O₂, thereby reducing it to O₂^•−. Without charge compensation, the membrane would depolarize to extreme positive voltages, around +200 mV, at which NADPH oxidase would cease functioning (16). Proton currents provide most of the charge compensation (17) and also diminish the cytosolic acidification resulting from oxidation of NADPH (18). Both charge compensation and regulation of cytosolic acidification are necessary to ensure the NADPH oxidase continues to function. Since this process does not take place in basophils, it is somewhat surprising to find proton channels to be expressed in these cells, especially at such high levels. However, they can regulate cytosolic pH upon activation (Fig. 2), as described by the DeCoursey lab recently (11), thus affecting those cellular processes that require pH regulation.

Figure 2. pH regulation by proton channels in basophils.

Average [H⁺]_i in basophils stimulated with 1 μg/ml anti-IgE in the absence (○) or presence of 100 μM Zn²⁺ (●) at ~30°C and imaged by using confocal microscopy and the shifted excitation and emission ratioing of fluorescence approach (SEER). The mean ± SEM of 25 control cells and 46 cells in Zn²⁺ is shown, with all data pairs after the star significantly different by Student’s t test (P < 0.05). Pseudocolor images indicating [H⁺]_i in trios of basophils from this experiment taken after stimulation with anti-IgE at the indicated time points are shown in two rows; the top row is control, the lower in the presence of Zn²⁺. Figure taken from Proceedings of the National Academy of Sciences of the United States of America (11).

Basophils, like tissue-resident mast cells, express the high-affinity receptor for IgE antibody, FcεRI. The high affinity binding of antigen-specific IgE to the FcεRI sensitizes basophils and mast cells to the specific antigen, such that a subsequent exposure to the antigen stimulates IgE-mediated activation of the cells. Activated basophils and mast cells secrete histamine, which is the primary chemical mediator contained in their cytoplasmic granules, as well as newly synthesized leukotriene C. The IgE mediated release of histamine and leukotriene C4 is the hallmark of an allergic reaction; however, basophils can also be activated by various IgE-independent stimuli. Recently, basophils have also become implicated in the initiation of Th2-type immune responses, such as allergic disorders and protective immunity to parasitic infections, through the production of large quantities of the Th2 cytokines, interleukins-4 and -13 (19).

In mast cells, histamine release is dependent on the activation of calcium-activated potassium channels, K_(Ca), presumably maintaining a driving force for Ca²⁺ entry, a key event in the activation of all leukocytes. In basophils, on the other hand, histamine release does not seem to depend on K_(Ca) channels (20), suggesting that alternative mechanisms might be in place to maintain a driving force for Ca²⁺ entry, such as the existence of outward proton currents. Three mechanisms of activating histamine release by basophils, PKC activation by phorbol myristate acetate (PMA), cross-linking of FcεRI-bound IgE by anti-IgE antibody and N-formyl-methionyl-leucyl-phenylalanine (fMLF), also activated proton channels. Although circumstantial, this evidence suggests that proton channel activity might be useful to the cell during histamine release. Consistent with this idea, inhibition of proton channels with Zn²⁺ caused inhibition of histamine release, highlighting an important role for proton channels during basophil activation (11). High concentrations of Zn²⁺, required to inhibit proton channels, could also result in inhibition of Ca²⁺ channels, impairing Ca²⁺ entry and consequently histamine release. However, Ca²⁺ influx in anti-IgE-stimulated basophils was not inhibited by up to 300 μM Zn²⁺, while proton currents were inhibited by 100 μM Zn²⁺ (11), indicating a specific effect of Zn²⁺ on proton channels.

Human basophils voltage-clamped in the perforated-patch configuration exhibited voltage-gated proton currents that resembled those observed in eosinophils, both qualitatively and quantitatively, at least in resting cells. At a symmetrical pH of 7, the proton conductance, g_H, activated during depolarizing voltage pulses above +20 mV, more rapidly at more positive voltages. Treatment with PMA, anti-IgE and N-formyl-methionyl-leucyl-phenylalanine (fMLF), another known agonist of histamine release (21), resulted in larger proton currents that activated more rapidly at each voltage. H⁺ current-voltage relationships revealed that the voltage at which the H⁺ current is first activated, V_threshold, was shifted 20 mV more negative after PMA stimulation and 11 mV after anti-IgE stimulation. Proton channel responses to both stimuli were qualitatively identical, although the anti-IgE responses were generally smaller.

The increase in proton current, also described as “enhanced gating mode”, was reversed by treatment with GF109203X or GFX, a well-characterized protein kinase C (PKC) inhibitor, although only partially in the case of anti-IgE stimulation. Enhanced gating in basophils was substantially less pronounced than in eosinophils, consistent with previous observations in cells lacking significant NADPH oxidase activity. The weaker enhanced gating in basophils, and similarly in CGD neutrophils, did not involve slower deactivation and the gH-V shift was only −20 mV, as opposed to −40 mV in eosinophils and neutrophils (22).

The enhanced gating mode is a modulation of proton channel activity regulated by phosphorylation of HVCN1 itself on residue Thr²⁹, as shown for proton channels in B cells (23). Phosphorylation is completely PKC-dependent, which explains why it could be completely reversed by treatment with GFX upon PMA stimulation. PKC, however, does not seem to be activated by anti-IgE treatment, as indicated by previous reports showing that histamine release is not affected by GFX treatment upon anti-IgE stimulation (21). It is interesting to note, however, that GFX is able to partially abrogate the increase in proton current, suggesting that the PKC-independent mechanisms regulating histamine release might also be responsible for the PKC-independent increase in proton channel activity. The kinetics of proton channel enhanced gating and degranulation suggest that despite a common upstream mediator of their activation, the proton channel response precedes degranulation.

An additional interesting point is that proton currents and their enhanced gating are not affected by the absence of Ca²⁺ in the bath solution or presence of a membrane permeable Ca²⁺ chelator such as 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetracetic acid, tetraacetoxymethyl ester (BAPTA-AM), suggesting that elevated [Ca²⁺]_i is not required, although it is essential for degranulation and histamine release after anti-IgE stimulation and activation with fMLF (11). Thus, it appears that proton channel activity and Ca²⁺ entry are regulated independently, although they can both affect histamine release. It is important to note that proton channels continue to function regardless of their enhanced gating status. They are simply more efficient when in enhanced gating mode, which might explain why the same signal (i.e. PKC activation) optimizes histamine release by switching proton channels to enhanced gating mode as well as initiating degranulation.

Confocal imaging of pH_i using the pH sensing SNARF dye in human basophils revealed that significant acid is produced during stimulation with anti-IgE (Fig. 2). Inhibition of proton channels with Zn²⁺ resulted in inhibition of histamine release that was accompanied by an increase in intracellular acidification following stimulation. This suggests that basophils undergo intracellular acidification upon activation and pH cannot be solely regulated by other H⁺ transporters, such as the Na⁺/H⁺ exchanger. It is possible to speculate that proton currents also help maintaining a negative membrane potential in activated basophils, supporting Ca²⁺ entry. However, this remains to be proven and further investigations will be required to corroborate this hypothesis. In summary, the action of proton channels, further improved by enhanced gating, can counterbalance intracellular acidification and potentially maintain a driving force for Ca²⁺ entry, allowing basophils to achieve maximal responses after stimulation.

HVCN1 in B lymphocytes

B lymphocytes constitute the arm of the adaptive immune system devoted to the generation of antibodies (also immunoglobulins), conferring protection against previously encountered antigens. Similar to phagocytic cells, B cells have the ability to endocytose antigens, although only when they bind to the immunoglobulin expressed on their surface, the B cell receptor (BCR). Antigen internalization leads to its digestion in lysosomes and, ultimately, presentation of antigen-derived peptides on major histocompatibility complex (MHC) class II molecules. Antigen internalization resembles phagocytosis of invading pathogens by phagocytes; similarly, it results in activation of the NADPH oxidase, the enzymatic complex responsible for ROS production, which is expressed by B cells as well as phagocytic cells (24, 25). B cells, however, do not engulf bacteria and therefore they do not need ROS to kill pathogens. Indeed, their ROS production is about 10-times smaller than that by neutrophils (24–26). The BCR-dependent ROS production is required to sustain signal transduction downstream of the BCR (12, 27). A requirement for ROS in BCR signal transduction was postulated initially by Michael Reth in the 1990s, when he showed that oxidizing agents were capable of activating B cells and resulted in phosphorylation of components of multiple downstream signaling pathways (27, 28). The resting state in B cells is the result of the balance between positive signals provided by kinases that are kept in check by negative signals provided by phosphatases, protein tyrosine phosphatases (PTP), in particular. The activity of PTP is much higher than the activity of kinases (it is less energy- and time-consuming to remove a phosphate group than to add it); therefore B cell activation could never take place unless an additional mechanism could block the activity of PTPs, at least temporarily. PTPs share a common feature in their catalytic site, a cysteine residue that resides in the signature sequence (I/V)HCXAGXXR(S/T)G. The close proximity to a positively charged amino acid results in the pK_a of the sulfhydryl group of the cysteine side chain, –SH, to be relatively low, rendering the –SH deprotonated at physiological pH (29). The resulting thiolate anion –S⁻, acts as a nucleophilic substrate that can attack the phosphorus of the PO₃ moiety bound to Tyr side-chain, leading to PO₄²⁻ release (29). Thiolate anion, however, is susceptible to oxidation, either reversible oxidation with weak oxidants or permanent oxidation with strong ones (30). This does not occur normally, due to the highly reducing environment of the cytosol. Nonetheless, the intracellular environment can become oxidizing, at least locally, following production of oxidants by the cells, as happens upon NADPH oxidase activation (27). In B cells, ROS generated by NADPH oxidase activity in the extracellular milieu or in the BCR-antigen-containing endolysosome can diffuse back into the cytosol and oxidize phosphatases recruited at the BCR complex, allowing optimal signal transduction (27). Indeed, treatment with antioxidants can impair lymphocyte activation (31). As shown in phagocytic cells, B cells require proton channels to sustain the activity of the NADPH oxidase, without proton channels, the enzymatic activity results in an intracellular accumulation of protons that causes excessive acidification as well as membrane depolarization, resulting in inhibition of NADPH oxidase and ultimately inhibition of ROS production (12).

B cells express proton channels at high levels, as observed by western blotting and patch-clamp experiments (12, 32). Proton currents in B cells are qualitatively similar to proton currents in neutrophils and eosinophils. Normalized for capacity, the mean H⁺ current density was 95 pA pF⁻¹ at +60 mV, pH_i 6 and pH_o 7.5 (32). On average, V_threshold was −20 mV, again, similar to proton currents in other leukocytes studied under the same conditions.

”Enhanced gating mode” has been reported in a B cell line transduced with exogenous HVCN1: it was possible to detect enhanced gating of proton channels in response to PMA, a function mediated by phosphorylation of Thr²⁹ in the cytoplasmic N-terminus domain of HVCN1 (23). It remains to be addressed whether BCR stimulation, which results in activation of PKC, can lead to enhanced gating of proton channels in primary B cells. Nonetheless, even without converting to the enhanced gating mode, proton channels in primary B cells are capable of compensating charge and pH in order to allow optimal NADPH oxidase activity.

Inhibition of proton channels with Zn²⁺ results in inhibition of ROS production in neutrophils, eosinophils, and monocytes (33, 34). In order to study the downstream consequences of proton channel inhibition in B cells, however, it was necessary to use a genetic mouse line in which the Hvcn1 gene had been knocked-out (KO) (12, 18, 35–37).

B cells from Hvcn1 KO mice appear to develop normally, an observation that correlated with the absence of Hvcn1 expression in B cell precursor cells (12). However, when mice were challenged with protein antigens, they showed impaired antibody responses, indicating that B cells were receiving a weaker stimulation in response to antigen recognition (12, 38, 39). Indeed, in vitro stimulated B cells appeared to respond normally at early time-points after BCR stimulation but were not able to sustain the activation of crucial signaling components such as spleen tyrosine kinase (Syk) and Akt (also known as protein kinase B or PKB) at later time-points. This was accompanied by a defect in the oxidation of a key protein tyrosine phosphatase, src homology 2 domain-containing tyrosine phosphatase, SHP-1. SHP-1 is recruited to the BCR complex upon activation, where it may be oxidized by locally-generated ROS (40). In the absence of Hvcn1, ROS production is not sustained, therefore SHP-1 oxidation is diminished, and the more active phosphatase can dephosphorylate its substrates such as the kinase Syk (Fig. 3) (12). It is interesting to note that B cells from Hvcn1 KO mice do not show complete absence of ROS production but rather a defect in its sustained generation. Accordingly, the initial activation of multiple BCR-dependent signaling pathways takes place normally in Hvcn1 KO B cells, whereas it cannot be sustained at later time-points. This could explain why different signaling pathways are not affected in the same way by the impaired ROS production; for example, Ca²⁺ mobilization and Erk activation are normal in Hvcn1 KO B cells. It is possible that different kinetics of activation explain this difference (41). It has recently emerged that the activation of BCR signaling pathways is also spatially regulated, with Erk being mainly activated while the BCR is at the plasma membrane and Akt being activated at the endolysosomes, once the BCR has been internalized (42). In light of these new results, it is possible to speculate that ROS production takes place only on the membrane of endolysosomes after the BCR has been internalized; therefore a defect in their production will affect signal initiated here (Akt); however it would not affect signal initiated at the membrane (Ca²⁺, Erk), as observed in Hvcn1 KO B cells. Further experiments to clarify where the NADPH oxidase is assembled and where it generates ROS within B cells would be required to clarify this point.

Figure 3. Schematic representation of HVCN1 in the context of BCR signaling.

Antigen binding to the BCR results in phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the Ig α/β heterodimer by Lyn, a src-family tyrosine kinase, creating docking sites for Syk (40). This serves to amplify BCR signaling by further recruitment and activation of Syk, which leads to PI3K activation, activation of Akt and increased energy 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 (40). SHP-1 dephosphorylates Syk, counterbalancing ITAM/Syk-mediated signal amplification. SHP-1 is inhibited by ROS, which oxidize a cysteine residue in the catalytic site of the enzyme. BCR stimulation results in ROS generated by the NADPH oxidase enzymatic complex, which transfers electrons across the plasma/endosome membrane to molecules of oxygen. The transfer of one electron results in the production of O₂^•− that combines with protons to form H₂O₂ and O₂, which freely diffuse through the membrane (2O₂^•− + 2H⁺→ H₂O₂ + O₂). ROS generate a localized oxidizing environment that leads to inhibition of SHP-1, which results in amplification of BCR signals. HVCN1 sustains NADPH oxidase activity through charge compensation and intracellular pH regulation; therefore, in the absence of HVCN1, the oxidizing environment cannot be maintained and this results in SHP-1 remaining more active, which diminishes BCR signal strength. Figure adapted from Nature Immunology (12).

The observation that Ca²⁺ signaling is not impaired in Hvcn1 KO B cells is in contrast to what Demaurex and coworkers observed in neutrophils deficient in HVCN1 (37, 43). Neutrophils evidently lack alternative pathways of charge compensation in the absence of proton channels, which results in depolarization that compromises the driving force for Ca²⁺ entry (37). It remains to be established why B cells do not seem to be affected in the same way. One possible explanation is the presence of K⁺ channels (44), which could provide the driving force for Ca²⁺ entry, although they do not appear sufficient to compensate for the loss of proton channels in sustaining NADPH oxidase activity, as shown in Hvcn1 KO B cells (12). A possible explanation is that NADPH oxidase activity in BCR-containing internalized endolysosomes can only be compensated by proton channels. Alternatively, it is possible that K⁺ channels may prevent excessive depolarization but (in contrast to proton channels) do not prevent the increased intracellular acidification that results from NADPH oxidase activity (2). In neutrophils, the NADPH oxidase-dependent acidification is sufficient to directly inhibit NADPH oxidase (18), as shown also in eosinophils (45). We have observed more pronounced acidification in BCR stimulated Hvcn1 KO B cells compared to controls (MC and Thomas DeCoursey, unpublished observation), however, further experiments would be required to prove unequivocally that pH dysregulation is the major factor responsible for impaired NADPH oxidase activity in Hvcn1 KO B cells. The defects observed in B cells in the absence of HVCN1 can be recapitulated by the defect in ROS production and downstream consequences on BCR signaling. It remains to be defined, however, whether the altered intracellular pH has additional consequences on B cell function in general and signal transduction in particular, which might contribute to the defective phenotype of Hvcn1 KO B cells.

HVCN1 in T lymphocytes

Proton currents have been described also in T lymphocytes, the adaptive immune cells responsible for cytokine production and killing of damaged cells, infected by viruses or carrying mutating genes. T cells are activated through their T cell receptor (TCR), when this recognizes peptide antigens bound to MHC class I and II molecules on antigen presenting cells or target cells. In contrast to B cells, resting T cells do not express NADPH oxidase (46, 47), although they appear to express NADPH oxidase components after stimulation (48) and a role for ROS in signaling has been reported also in T cells (31, 49). It is believed that ROS that affect T cell signaling in the initial stages of T cell activation might be provided extracellularly, by other cells in the extracellular environment (46).

In their study published in 2002, Eder and coworkers evaluated proton currents in primary T cells and B cells for the first time (Fig. 4) (32). They found proton currents in T cells to be much smaller than in B cells, 70 times smaller, while current density was over 100 times smaller. However, after activation for 24h with PMA, proton current density increased 8-fold, suggesting that T cells do not require proton currents during the initial phases of cell activation, as B cells do, but instead they might play a role in activated cells. The time frame of proton channels expression after stimulation appears consistent with the expression of NADPH oxidase subsequently reported by Jackson et al. (48). As observed in B cells, activated T cells might utilize proton currents to sustain ROS production by the NADPH oxidase, as well as relieve intracellular acidification due to metabolic processes taking place in the cell. In contrast to resting T cells, activated cells undergo a very rapid clonal expansion, requiring a sharp increase in metabolic processes to ensure high levels of protein, RNA and DNA synthesis, as well as provision of ATP to sustain them (50). Therefore, their risk of intracellular acidification is higher and acidification could inhibit important processes such as IL-2 production (51). Furthermore, it has been reported that following activation with mitogens, pH_i of T cells increases (51, 52); hence proton channels might constitute one of the components that helps stabilizing the more alkaline pH_i. It is important to note, however, that the amplitude of proton currents in activated T cells remains small, suggesting that their role in regulating T cell function might be a minor one.

Figure 4. H⁺ currents in human peripheral blood lymphocytes.

Superimposed H⁺ currents in a CD19⁺ B lymphocyte (A) and a CD3⁺ T lymphocyte (B) elicited by identical families of 4 s voltage pulses from the holding potential of −60 mV to potentials between −40 and +60 mV, both at pH_i 6.0 and pH_o 7.5. C, current-voltage relationships for H⁺ currents at the end of the pulses shown in A and B (●, B lymphocyte; ■, T lymphocyte). D, average (± SEM) H⁺ current densities in lymphocyte subpopulations. H⁺ current densities were calculated from the H⁺ current measured at the end of a 4 s pulse to +60 mV, and normalized to the capacity in each cell. Data are from 63 human B lymphocytes, 46 human T lymphocytes, and 52 Jurkat T cells at pH_i 6.0 and pH_o 7.5. Figure taken from the Journal of Physiology (32).

Conclusions

Proton channels have been described in phagocytic and non-phagocytic cells of the immune system and appear to play an important role in these cells. In cells where proton channel expression is accompanied by NADPH oxidase expression, such as B cells and activated T cells, they appear to have similar roles to the ones described for phagocytic cells: sustaining NADPH oxidase activity, possibly through charge compensation, possibly through intracellular pH regulation or maybe both. In basophils, which lack NADPH oxidase expression, proton channels seem to regulate intracellular pH and possibly maintain a driving force for Ca²⁺ entry, although further experiments will be required to clarify their role in more details.

The identification of the HVCN1 gene sparked a renewed interest in proton channels in recent years; this will probably result in further characterization of the electrophysiological properties of the channel, as well as the emergence of additional roles played in other cell types not yet investigated. It is certainly an interesting time to be studying voltage-gated proton channels.