Hv1 Expression Research Articles

Abstract 2060: Hv1 Contributes To Atherosclerosis

Atherosclerosis is a complex process that involves the interaction between oxidized low-density lipoprotein (oxLDL) and macrophage. It is an inflammatory disease of the large artery driven by macrophage activation and infiltration. Through scavenger receptors, macrophages can uptake oxLDL and other lipids, leading to foam cell formation and the progression of atherosclerosis. Hv1 proton channel is highly expressed in phagocytes and promotes NADPH oxidase-derived ROS (reactive oxygen species) production. So far, most of the research on Hv1 has been focused on immune, reproductive, and neurological diseases. However, the role of Hv1 in cardiovascular diseases, such as arteriosclerosis, remains unclear. To explore the potential roles of Hv1 in atherosclerosis, we detected the expression of Hv1 in atherosclerotic lesions. It was shown that Hv1 is abundantly expressed in human and murine atherosclerotic lesions and highly expressed in macrophages in the aortic root lesions. In addition, Hv1 knockout mice fed with a high-fat western diet exhibit remarkably decreased atherosclerotic plaques compared with control mice. Moreover, deficiency of Hv1 in macrophages impedes macrophage phagocytosis and foam cell formation essential for atherosclerosis development, and Hv1 enhances macrophage ROS production associated with the progression of atherosclerosis. Our results suggested that Hv1 is a pro-atherosclerotic factor and contributes to the pathogenesis of atherosclerosis. The research identified a new function of the Hv1 channel in human diseases and may present a novel target against atherosclerosis and atherosclerosis-related diseases.

Traumatic brain injury-induced inflammatory changes in the olfactory bulb disrupt neuronal networks leading to olfactory dysfunction

Approximately 20–68% of traumatic brain injury (TBI) patients exhibit trauma-associated olfactory deficits (OD) which can compromise not only the quality of life but also cognitive and neuropsychiatric functions. However, few studies to date have examined the impact of experimental TBI on OD. The present study examined inflammation and neuronal dysfunction in the olfactory bulb (OB) and the underlying mechanisms associated with OD in male mice using a controlled cortical impact (CCI) model. TBI caused a rapid inflammatory response in the OB as early as 24 h post-injury, including elevated mRNA levels of proinflammatory cytokines, increased numbers of microglia and infiltrating myeloid cells, and increased IL1β and IL6 production in these cells. These changes were sustained for up to 90 days after TBI. Moreover, we observed significant upregulation of the voltage-gated proton channel Hv1 and NOX2 expression levels, which were predominantly localized in microglia/macrophages and accompanied by increased reactive oxygen species production. In vivo OB neuronal firing activities showed early neuronal hyperexcitation and later hypo-neuronal activity in both glomerular layer and mitral cell layer after TBI, which were improved in the absence of Hv1. In a battery of olfactory behavioral tests, WT/TBI mice displayed significant OD. In contrast, neither Hv1 KO/TBI nor NOX2 KO/TBI mice showed robust OD. Finally, seven days of intranasal delivery of a NOX2 inhibitor (NOX2ds-tat) ameliorated post-traumatic OD. Collectively, these findings highlight the importance of OB neuronal networks and its role in TBI-mediated OD. Thus, targeting Hv1/NOX2 may be a potential intervention for improving post-traumatic anosmia.

Role of Hv1 channel in cancer immunoprotection and malignancy.

Hv1 is linked to immunosuppression mediated by MDSC through the modulation of function of NOX2. The carcinogenesis process includes several changes to local temperature, pain and, especially important, pH. We studied the relation of Hv1 activity within melanoma in murine model by using, flow-cytometry, biochemistry and patch-clamp in whole cell configuration. In this work, we demonstrated that the inhibition of the Hv1 by its canonical blockers (Zn+2 and Cl-GBI) reduces the ROS production on MDSC which in turn decreases immunosuppression around the tumor mass. By inducing the growth of melanoma B16F0 in mice by inoculation and then treating the animals with the Hv1 inhibitors we found a dramatic reduction on tumor mass volume (∼35%). These results suggest that the inhibition of Hv1 activity promotes the immune response within the model, in turn, reducing the growth rate of the tumoral mass. On the other hand, MDSC present an increment on Hv1 expression through their differentiation process maximizing when reaching its final stages as dendritic cells or neutrophiles. By using non-stationary noise analysis of Hv1 channel we calculated the number of channels present in the cells when reaching their MDSC stage to 9 channel/µm. However, when comparing samples of cancer with different malignancy (HeLa vs B16F0), the levels of expression of Hv1 shows a stark difference. Our result suggest that the degrees of malignancy seem to be directly proportional to the expression of Hv1. Setting Hv1 as both a prognosis marker for cancer and a potential therapeutic target

TMIC-09. HV1 PROTON CHANNELS PROMOTE MYELOID INFILTRATION AND GLIOMA PROGRESSION

Abstract Myeloid cells comprising up to 50% of total tumor mass in glioblastoma (GBM) contribute to tumor progression and immunosuppression. Restraining glioma-induced myeloid cell infiltration increases functional T cells within the tumors, improves the efficacy of checkpoint blockade, and significantly extends mouse survival. Here, we focus on an ion channel, voltage-gated proton channel Hv1, which is mainly expressed in myeloid cells and shapes the physiological functions of myeloid cells. Our bioinformatics analysis demonstrated that elevated Hv1 expression in the tumor mass correlates with poor disease prognosis in patients. Using the leading immunocompetent model of GBM in mice, we showed that Hv1 knockout mice (Hv1-/-) exhibit slower glioma progression and prolonged survival. Notably, in vivo two-photon imaging, immunofluorescence, and full-spectrum flow cytometry revealed that Hv1-/- mice have reduced monocyte/macrophage infiltration, higher frequency of MHCII+ among all infiltrating myeloid cells, and increased PD-1+CD4+ T cells in the tumor-burdened hemisphere. As a result, we combined anti-PD-1 treatment with Hv1 knockout in the middle stage of glioma, and found that approximately 30% of Hv1-/- mice were cured. Together, our results demonstrated that Hv1 regulates myeloid infiltration and could be a novel therapeutic target for the treatment of GBM.

Hv1 proton channels control myeloid landscape and promote glioma progression

Abstract Myeloid cells comprising up to 50% of total tumor mass in glioblastoma (GBM) contribute to tumor progression and immunosuppression. Restraining glioma-induced myeloid cell infiltration increases functional T cells within the tumors, improves the efficacy of checkpoint blockade, and significantly extends mouse survival. Here, we focus on an ion channel, voltage-gated proton channel Hv1, which is mainly expressed in myeloid cells and shapes the physiological functions of myeloid cells. Our bioinformatics analysis demonstrated that elevated Hv1 expression in the tumor mass correlates with poor disease prognosis in patients. Using the leading immunocompetent model of GBM in mice, we showed that Hv1 knockout mice (Hv1−/−) exhibit slower glioma progression and prolonged survival. Using in vivo two-photon imaging, we found that myeloid cells infiltrate into tumor mass since early time points and have intimate interaction with tumor cells. Notably, by full-spectrum flow cytometry, we found that Hv1−/− mice have reduced monocyte/macrophage infiltration, and increased PD-1+CD4+ T cells in the tumor-burdened hemisphere. As a result, we combined anti-PD-1 treatment with Hv1 knockout in the middle stage of glioma, and found that approximately 30% of Hv1−/− mice were cured. Together, our results demonstrated that Hv1 controls myeloid infiltration and could be a novel therapeutic target for the treatment of GBM. Supported by R01NS110825, R01NS088627

Inhibiting Hv1 channel in peripheral sensory neurons attenuates chronic inflammatory pain and opioid side effects.

Both opioids and nonsteroidal anti-inflammatory drugs (NSAIDS) produce deleterious side effects and fail to provide sustained relief in patients with chronic inflammatory pain. Peripheral neuroinflammation (PN) is critical for initiation and development of inflammatory pain. A better understanding of molecular mechanisms underlying PN would facilitate the discovery of new analgesic targets and the development of new therapeutics. Emerging evidence suggests that peripheral sensory neurons are not only responders to painful stimuli, but are also actively engaged in inflammation and immunity, whereas the intrinsic regulatory mechanism is poorly understood. Here we report the expression of proton-selective ion channel Hv1 in peripheral sensory neurons in rodents and humans, which was previously shown as selectively expressed in microglia in mammalian central nervous system. Neuronal Hv1 was up-regulated by PN or depolarizing stimulation, which in turn aggravates inflammation and nociception. Inhibiting neuronal Hv1 genetically or by a newly discovered selective inhibitor YHV98-4 reduced intracellular alkalization and ROS production in inflammatory pain, mitigated the imbalance in downstream SHP-1-pAKT signaling, and also diminished pro-inflammatory chemokine release to alleviate nociception and morphine-induced hyperalgesia and tolerance. Thus, our data reveal neuronal Hv1 as a novel target in analgesia strategy and managing opioids-related side effects.

Amide proton transfer (APT) imaging-based study on the correlation between brain pH and voltage-gated proton channels in piglets after hypoxic-ischemic brain injury.

The normal regulation of brain pH is particularly critical for protein structure and enzymatic catalysis in the brain. This study aimed to investigate the regulation mechanism of brain pH after hypoxic-ischemic brain injury (HIBI) through the combination of amide proton transfer (APT) imaging, the analysis of brain pH levels, and the analysis of voltage-gated proton channel (Hv1) expression in piglets with HIBI. A total of 59 healthy piglets (age range, 3-5 days after birth; body weight, 1-1.5 kg) were selected. Six piglets were excluded due to death, modeling failure, or motion artifacts, leaving a total of 10 animals in the control group and 43 animals in the HIBI model group. At different time points (0-2, 2-6, 6-12, 12-24, 24-48, and 48-72 hours) after HIBI, brain pH, Hv1 expression, and APT values were measured and analyzed. The statistical analysis of data was performed using the independent samples t-test, analysis of variance, and Spearman rank correlation analysis. A P value less than 0.05 indicated statistical significance. As shown by the immunofluorescent staining results after HIBI, Hv1 protein expression in the basal ganglia reached a peak value at 0-2 hours, with a statistically significant difference between 0-2 hours and other time points (P<0.001). In piglets, the APT value reached a trough at 0-2 hours after HIBI, and subsequently, it gradually increased, and there was a significant difference between the control group and all HIBI model subgroups (P<0.001) except for the 2-6 hours subgroup (P=0.602). Brain pH decreased after HIBI and reached a trough at 0-2 hours, then gradually increased. Hv1 protein expression, pH, and APT values were all correlated (P<0.001). After HIBI, values of brain pH, APT, and the expression of Hv1 changed over time and had a linear correlation. This suggests that there was a shift in brain hydrogen ions (H+) in the neural network and a change in brain pH after hypoxic-ischemic (HI) injury.

Functions and Mechanisms of the Voltage-Gated Proton Channel Hv1 in Brain and Spinal Cord Injury.

The voltage-gated proton channel Hv1 is a newly discovered ion channel that is highly conserved among species. It is known that Hv1 is not only expressed in peripheral immune cells but also one of the major ion channels expressed in tissue-resident microglia of the central nervous systems (CNS). One key role for Hv1 is its interaction with NADPH oxidase 2 (NOX2) to regulate reactive oxygen species (ROS) and cytosolic pH. Emerging data suggest that excessive ROS production increases and requires proton currents through Hv1 in the injured CNS, and manipulations that ablate Hv1 expression or induce loss of function may provide neuroprotection in CNS injury models including stroke, traumatic brain injury, and spinal cord injury. Recent data demonstrating microglial Hv1-mediated signaling in the pathophysiology of the CNS injury further supports the idea that Hv1 channel may function as a key mechanism in posttraumatic neuroinflammation and neurodegeneration. In this review, we summarize the main findings of Hv1, including its expression pattern, cellular mechanism, role in aging, and animal models of CNS injury and disease pathology. We also discuss the potential of Hv1 as a therapeutic target for CNS injury.

Respiratory syncytial virus infection of microglia exacerbates SH-SY5Y neuronal cell injury by inducing the secretion of inflammatory cytokines: A Transwell in vitro study.

Objective(s):To elucidate the mechanism of Respiratory Syncytial Virus (RSV) infection and central neuronal disease and to understand the role of microglia in neuronal injuries during RSV infection.Materials and Methods: The effects of RSV and the cytokines produced by RSV-infected CHME-5 microglial cells on SY5Y neuronal cells were evaluated based on an in vitro Transwell coculture system. Five treatment groups were established in this study, including the normal control SY5Y group, RSV+SY5Y infection group, (cytokine+CHME-5)+SY5Y Transwell group, (RSV+CHME-5)+SY5Y Transwell group, and (RSV+cytokine+CHME-5)+SY5Y Transwell group. The morphological and physical alterations in SY5Y cells and their synapses were analyzed by confocal microscopy. The mRNA and protein expression levels of TLR3/RIG-I, as well as the expression of Hv1, in microglia were measured by qRT-PCR and Western blot assays. In addition, the apoptosis ratio of neuronal cells was determined by flow cytometry.Results:RSV infection activated the protein expression of Hv1 protein in microglia in vitro (P<0.05), induced morphological changes in SY5Y cells, lengthened synapses (73.36±0.12 μm vs 38.10±0.11 μm), simultaneously activated TLR3 and RIG-I protein expression (P<0.05), upregulated the secretion of the inflammatory cytokines TNF-α, IL-6, and IL-8 (P<0.01), and increased the apoptosis rate of SY5Y cells (P<0.01).Conclusion:The results demonstrate that RSV infection of microglia can induce SY5Y neuronal cell injury and stimulate apoptosis through inflammatory cytokine release.

The voltage-gated proton channel Hv1 plays a detrimental role in contusion spinal cord injury via extracellular acidosis-mediated neuroinflammation

Tissue acidosis is an important secondary injury process in the pathophysiology of traumatic spinal cord injury (SCI). To date, no studies have examined the role of proton extrusion as mechanism of pathological acidosis in SCI. In the present study, we hypothesized that the phagocyte-specific proton channel Hv1 mediates hydrogen proton extrusion after SCI, contributing to increased extracellular acidosis and poor long-term outcomes. Using a contusion model of SCI in adult female mice, we demonstrated that tissue pH levels are markedly lower during the first week after SCI. Acidosis was most evident at the injury site, but also extended into proximal regions of the cervical and lumbar cord. Tissue reactive oxygen species (ROS) levels and expression of Hv1 were significantly increased during the week of injury. Hv1 was exclusively expressed in microglia within the CNS, suggesting that microglia contribute to ROS production and proton extrusion during respiratory burst. Depletion of Hv1 significantly attenuated tissue acidosis, NADPH oxidase 2 (NOX2) expression, and ROS production at 3 d post-injury. Nanostring analysis revealed decreased gene expression of neuroinflammatory and cytokine signaling markers in Hv1 knockout (KO) mice. Furthermore, Hv1 deficiency reduced microglia proliferation, leukocyte infiltration, and phagocytic oxidative burst detected by flow cytometry. Importantly, Hv1 KO mice exhibited significantly improved locomotor function and reduced histopathology. Overall, these data suggest an important role for Hv1 in regulating tissue acidosis, NOX2-mediated ROS production, and functional outcome following SCI. Thus, the Hv1 proton channel represents a potential target that may lead to novel therapeutic strategies for SCI.

Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells.

Expression of the voltage gated proton channel (Hv1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of HV1 expression in 3 out of 8 “triple negative” breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (knock-down; KD) and by eliminating HV1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 Knock-out cells did not show reduced migration or H2O2 release. HV1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in HV1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although HV1 KO and KD affect certain cellular mechanisms differently, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.

Loss of the voltage-gated proton channel Hv1 decreases insulin secretion and leads to hyperglycemia and glucose intolerance in mice

Insulin secretion by pancreatic islet β-cells is regulated by glucose levels and is accompanied by proton generation. The voltage-gated proton channel Hv1 is present in pancreatic β-cells and extremely selective for protons. However, whether Hv1 is involved in insulin secretion is unclear. Here we demonstrate that Hv1 promotes insulin secretion of pancreatic β-cells and glucose homeostasis. Hv1-deficient mice displayed hyperglycemia and glucose intolerance because of reduced insulin secretion but retained normal peripheral insulin sensitivity. Moreover, Hv1 loss contributed much more to severe glucose intolerance as the mice got older. Islets of Hv1-deficient and heterozygous mice were markedly deficient in glucose- and K+-induced insulin secretion. In perifusion assays, Hv1 deletion dramatically reduced the first and second phase of glucose-stimulated insulin secretion. Islet insulin and proinsulin content was reduced, and histological analysis of pancreas slices revealed an accompanying modest reduction of β-cell mass in Hv1 knockout mice. EM observations also indicated a reduction in insulin granule size, but not granule number or granule docking, in Hv1-deficient mice. Mechanistically, Hv1 loss limited the capacity for glucose-induced membrane depolarization, accompanied by a reduced ability of glucose to raise Ca2+ levels in islets, as evidenced by decreased durations of individual calcium oscillations. Moreover, Hv1 expression was significantly reduced in pancreatic β-cells from streptozotocin-induced diabetic mice, indicating that Hv1 deficiency is associated with β-cell dysfunction and diabetes. We conclude that Hv1 regulates insulin secretion and glucose homeostasis through a mechanism that depends on intracellular Ca2+ levels and membrane depolarization.

Loss of voltage-gated proton channel Hv1 leads to diet-induced obesity in mice

ObjectiveThe voltage-gated proton channel Hv1 has been proposed to mediate NADPH oxidase (NOX) function by regulating intracellular pH during respiratory bursts. In our previous work, we showed that Hv1 is...

Ion channel gene expressions in infertile men: A case-control study

Infertility is described as not receiving pregnancy despite unprotected and regular sexual intercourse in a 1 yr period. It is detected by 15% of the couples. Male and female factor in the etiology may be detected in similar rates. The present study aims to investigate ion channel gene expression in semen samples of infertile male compared with fertile men. A total of 150 men who applied to the urology clinic due to infertility were divided into five equal groups: asthenozoospermia, oligozoospermia, oligoasthenoteratozoospermia, teratozoospermia, and normozoospermia (control). All paticipants were evaluated with Cation Channel Spermia (CatSper) 1, 2, 3, 4, Proton Voltage Gated Ion Channel1 (Hv1), Potassium Channel Subfamily U1 (KCNU1), and transmembrane protein (TMEM16A) gene expression in semen samples. "CatSper1, 4, HV1, KCNU1, and TMEM16A gene expression were detected higher in the oligozoospermia group compared to the controls. CatSper1, 2, 3, 4, KCNU1, and TMEM16A gene expression in the asthenozoospermia group and CatSper1, 2, 3, 4, KCNU1, and TMEM16A gene expression in the teratozoospermia group were detected lower compared to the controls. CatSper1, 4, HV1, and TMEM16A gen expression were higher in the oligoasthenoteratozoospermia men than the controls while CatSper3 gen expression was detected as lower." It was detected that these ion channels have an effect on sperm progressive motility and morphology. It may be considered that mutations in these ion channels may result in infertility.

The voltage-gated proton channel Hv1 is expressed in pancreatic islet β-cells and regulates insulin secretion

The voltage-gated proton channel Hv1 is a potent acid extruder that participates in the extrusion of the intracellular acid. Here, we showed for the first time, Hv1 is highly expressed in mouse and human pancreatic islet β-cells, as well as β-cell lines. Imaging studies demonstrated that Hv1 resides in insulin-containing granules in β-cells. Knockdown of Hv1 with RNA interference significantly reduces glucose- and K+-induced insulin secretion in isolated islets and INS-1 (832/13) β-cells and has an impairment on glucose- and K+-induced intracellular Ca2+ homeostasis. Our data demonstrated that the expression of Hv1 in pancreatic islet β-cells regulates insulin secretion through regulating Ca2+ homeostasis.

Proton Channels are Present in Cell Membranes of the Breast Cancer Cell Line MDA MB 231 and Affect Recovery from an Acid Load

Voltage gated proton channels (HV1) have been implicated in late stage breast cancer (Wang et al, 2012. J. Biol. Chem. 287:13877), where HV1 expression correlated with tumor size and poor prognosis. HV1 knockdown reduced cell proliferation and migration as well as matrix metalloprotease release. However, the presence of functional HV1 on cancer cell membranes has not been demonstrated and the mechanism by which they affect the function of these cells has not been elucidated.Here we show the definitive presence of functional HV1 on the membranes of MDA MB 231 cells, a highly metastatic triple negative cell line. We performed patch clamp experiments on these cells and were able to detect bona fide voltage- and pH-gated channels that were perfectly selective for protons. The membrane density of the channels in these cells was recorded as 3.5 pA/pF which is roughly 5-fold lower than the 15 pA/pF displayed in neutrophils. In order to show that HV1 expresses at a level sufficient to impact pH regulation within these cells, we acid loaded them using the ammonium prepulse technique and monitored pHi recovery utilizing SEER with SNARF-1. Inhibiting HV1 with 1 mM Zn2+ slowed recovery from an acid load by 3-fold, demonstrating that the expression of HV1 on these cells affects pH regulation in these cells.We conclude that one mechanism by which HV1 may influence the pathophysiology of breast cancer is by improving the ability of breast cancer cells to regulate their internal pH.Supported by the Bears Care and the Brian Piccolo fund.

Characterization and Subcellular Localization of Hv1 in Lingulodinium Polyedrum Confirms its Role in Bioluminescence

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel that opens in response to depolarizing voltage (HV1) across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thus causing the pH drop that triggers the light flash. RNA-Seq data from several luminescent dinoflagellate species provided candidate HV1 genes. When expressed in mammalian cells, the predicted HV1 from Lingulodinium polyedrum displays the hallmark properties of bona fide proton channels, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. RT-PCR and Western blotting confirm expression of HV1 in L. polyedrum. Fluorescence confocal microscopy of L. polyedrum cells stained with antibodies to luminescence proteins luciferase and luciferin binding protein and to HV1 reveal structures consistent with HV1's proposed function in bioluminescence.[Supported by NSF: MCB-1242985, NIH: GM102336, ⋯]

Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was ∼10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was ∼1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only ∼1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface.

Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases

Production of ROS and maintenance of an appropriate pH within the lumen of neutrophil and macrophage phagosomes are important for an effective immune response. Hv1 proton channels sustain ROS production at the plasma membrane, but their role in phagosomes is not known. Here, we tested whether Hv1 channels regulate the pHp and sustain phagosomal ROS production in neutrophils and macrophages. The presence of Hv1 channels on phagosomes of human neutrophils and mouse macrophages was confirmed by Western blot and immunostaining. Phagosomal ROS production, measured with OxyBurst-coupled targets, was reduced in neutrophils and macrophages isolated from Hv1-deficient mice. Ratiometric imaging of FITC-coupled targets showed that phagosomes acidified more slowly in Hv1-deficient macrophages and transiently alkalinized when the V-ATPase was inhibited. In WT neutrophils, 97% of phagosomes remained neutral 30 min after particle ingestion, whereas 37% of Hv1-deficient phagosomes were alkaline (pH>8.3) and 14% acidic (pH<6.3). The subpopulation of acidic phagosomes was eliminated by V-ATPase inhibition, whereas NOX inhibition caused a rapid acidification, independently of Hv1 expression. Finally, V-ATPase accumulation on phagosomes was inversely correlated to intraphagosomal ROS production in neutrophils. These data indicate that Hvcn1 ablation deregulates neutrophil pHp, leading to alkalinization in phagosomes with residual ROS production or to the early accumulation of V-ATPase on phagosomes that fail to mount an oxidative response. Hv1 channels therefore differentially regulate the pHp in neutrophils and macrophages, sustaining rapid acidification in macrophage phagosomes and maintaining a neutral pH in neutrophil phagosomes.

Human Voltage-Gated Proton Channel Hv1: A New Potential Biomarker for Diagnosis and Prognosis of Colorectal Cancer

Solid tumors exist in a hypoxic microenvironment, and possess high-glycolytic metabolites. To avoid the acidosis, tumor cells must exhibit a dynamic cytosolic pH regulation mechanism(s). The voltage-gated proton channel Hv1 mediates NADPH oxidase function by compensating cellular loss of electrons with protons. Here, we showed for the first time, that Hv1 expression is increased in colorectal tumor tissues and cell lines, associated with poor prognosis. Immunohistochemistry showed that Hv1 is strongly expressed in adenocarcinomas but not or lowly expressed in normal colorectal or hyperplastic polyps. Hv1 expression in colorectal cancer is significantly associated with the tumor size, tumor classification, lymph node status, clinical stage and p53 status. High Hv1 expression is associated significantly with shorter overall and recurrence-free survival. Furthermore, real-time RT-PCR and immunocytochemistry showed that Hv1 is highly expressed in colorectal cancer cell lines, SW620, HT29, LS174T and Colo205, but not in SW480. Inhibitions of Hv1 expression and activity in the highly metastatic SW620 cells by small interfering RNA (siRNA) and Zn2+ respectively, markedly decrease the cell invasion and migration, restraint proton extrusion and the intracellular pH recovery. Our results suggest that Hv1 may be used as a potential biomarker for diagnosis and prognosis of colorectal carcinoma, and a potential target for anticancer drugs in colorectal cancer therapy.