Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis, and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH i regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-␦ phosphorylation sites in the human voltage-gated proton channel, H V 1 (Hvcn1), Thr 29 and Ser 97 , both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-␦. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H V 1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H V 1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr 29 is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.Voltage-gated proton channels enable sustained superoxide anion (O 2 . ) production by NADPH oxidase during the respira- efflux through open voltage-gated proton channels (1-6). The activities of NADPH oxidase and voltage-gated proton channels are coordinated in several ways. The depolarization and pH i decrease resulting from NADPH oxidase activity both directly promote proton channel opening. In addition, interventions that activate NADPH oxidase profoundly enhance the gating properties of proton channels (3, 7). This "enhanced gating mode" consists of four changes in proton channel properties, each of which increases the likelihood of channel opening under any given set of conditions. The channels open faster (smaller activation time constant, act ) 3 and close more slowly (larger deactivation time constant, tail ), display increased maximum proton conductance (g H,max ), and manifest a 40-mV hyperpolarizing shift of the entire proton conductance-voltage relationship (g H -V). The enhanced gating mode improves the efficiency of NADPH oxidase by minimizing the depolarization required to open enough proton channels to fully compensate the electrical consequences of NADPH oxidase activity (i.e. the electron current) (5). Depolarization directly inhibits NADPH oxidase (4, 8).The enhanced gating mode is induced by PMA, an activator of PKC, and is prevented and at least partially reversed by the PKC inhibitor GFX (9, 10). Although these results suggest regulation by PKC phosphorylation, they do not clarify whether the target of PKC is an accessor...

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1 S ) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1 S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1 L , Thr 29 . Furthermore, the association of HVCN1 S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1 S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1 S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies. is a small protein that conducts protons across membranes selectively (1, 2) and in a regulated manner. Previously, we described its function in B lymphocytes, where proton channels sustain B-cell receptor (BCR) signaling via regulation of reactive oxygen species production by the NADPH oxidase enzyme complex (3). In addition, we found HVCN1 to be directly associated with the BCR. Upon receptor stimulation, the BCR and HVCN1 were cointernalized to late endosomal/lysosomal organelles called "MIICs," or MHC class II-containing compartments, where antigens bound to the BCR are digested into small peptides and loaded onto MHC class II molecules for presentation to T cells (3).HVCN1 is expressed not only by normal but also by malignant B cells, such as those in chronic lymphocytic leukemia (CLL) (3). CLL cells are characterized by their reliance on BCR signaling for survival and growth (4), so it is possible that they maintain or upregulate HVCN1 expression to sustain their growth. Other tumor cells, such as those in breast (5) and colorectal cancer (6), have been found to rely on HVCN1 for survival. In these tumor cells, proton channels prevent excessive acidification of the cytoplasm and allow increased cell migration. In malignant B cells, HVCN1 may regulate intracellular pH and at the same time sustain BCR signaling. However, its precise roles remain to be elucidated.We show here that CLL cells and other B-cell lines specifically express higher levels of a shorter isoform of HVCN1, HVCN1 S . We identified the existence of two distinct isoforms of relatively similar size when immunoblotting B-cell lysates with an HVCN1-specific antibody (3). HVCN1 S ...

epithelium prone to infection and in intestine leads to obstruction. Patients homozygous for F508del, have a tremendous variation in the severity of disease. Recent Genome-wide association studies indicate that this variation is due to presence of modifier genes, with SLC6A14 as the top modifier (Sun et.al.,2012). SLC6A14 is a Naþ/Cl-dependent cationic/neutral amino-acid transporter on the surface of lung and colonic epithelium. As both transporters are expressed apically, we hypothesized that SLC6A14 would modify the fluid secretory capacity of the epithelium. So in collaboration with TCP, we generated a SLC6A14 knock-out mouse. We can measure in-vivo fluid secretion in mice, using an intestinal closed-loop assay. SLC6A14 knock-out mice exhibited a decrease in cAMP stimulated fluid secretion mediated via CFTR relative to Wt control. To explore the mechanism by which this modification occurs, we utilized a BHK heterologous expression system, overexpressing CFTR and SLC6A14. Interestingly, the functional interaction can be recapitulated in this system, suggesting that it not tissue-type dependent. Preliminary biochemical and anion-flux studies support the hypothesis that SLC6A14 does not affect the processing or stability of Wt or F508del-CFTR proteins rather it augments the activity of these channel proteins once localized to the cell surface. Future studies will focus on understanding if this augmentation is related to modification of CFTR's phosphorylation dependent gating. These results show a positive impact of SLC6A14 on CFTR channel function and fluid secretion, providing an alternative drug target for CF patients. 96-PlatEnhanced Activation of an Amino-Terminally Truncated Isoform of Voltage-Gated Proton Channel HVCN1 Enriched in Malignant B cells

HVCN1 is a highly-conserved voltage-gated proton channel. Voltage-gated proton currents have been recorded in lymphocytes but their functions in B cells remain unknown. We isolated HVCN1 in a proteomic survey of plasma membrane proteins in mantle cell lymphoma (MCL) in leukemic phase. In normal lymphocytes, HVCN1 expression was restricted to the B-cell lineage; HVCN1 was highly expressed in mantle zone cells but down-regulated in germinal center (GC) cells undergoing receptor affinity maturation and class-switch recombination (CSR). Highest level expression was also observed in Chronic Lymphocytic Leukemia (CLL) cells from the peripheral blood. In MCL tumors, HVCN1 was expressed in circulating cells but absent from involved lymph nodes, whereas in diffuse large B cell lymphoma (DLBCL), its expression correlated with cases with a low proliferation index. Thus, in both primary and neoplastic B cells, HVCN1 expression appears to be associated with a non-proliferative phenotype. In human primary resting B cells and B cell lines, HVCN1 directly interacted with the B cell receptor (BCR) complex, as shown by Igβ and HVCN1 reciprocal immunoprecipitation experiments. We also found by confocal microscopy and subcellular fractionation, that upon BCR engagement the channel was internalized with the antigen receptor and the two proteins co-migrated to the endo-lysosomal, MHC class II (MHC-II) containing compartments (MIICs). When overexpressed in a hen egg lysozyme (HEL)-specific B cell clone, LK35.2, HVCN1 showed a basal phosphorylation which increased with HEL stimulation. The increased phosphorylation corresponded to an increase in proton conductance, termed “enhanced gating mode” and it was PKC dependent. We then asked whether HVCN1 over-expression could influence MHC II antigen presentation and if the effect could be mediated by changes in MIICs pH. Indeed, presentation of HEL peptides to a T cell clone was impaired in LK35.2 and A20 D1.3 cells, where HVCN1 had been re-introduced; effect was stronger for plate-bound antigen than for soluble antigen. The reduced antigen presentation was accompanied by an increase in endo-lysosomal pH, from pH4.9 ± 0.2 to 6.3 ± 0.1 (which may reflect HVCN1 channel-mediated proton flux out of the organelles), as measured with an anti-IgM antibody conjugated to a pH sensitive dye in HVCN1 over-expressing cells. Evidently, the presence of HVCN1 leads to increased endo-lysosomal pH, consistent with H+ current from the lysosomal compartment into the cytosol. Hence, active antigen presenting cells, like GC cells, might down-regulate HVCN1 expression to maximize the effect of antigen presentation. In order to investigate the role of HVCN1 in vivo, we used a HVCN1-deficient mouse line generated by genetrap insertion. These mice showed no obvious changes in numbers or composition of B-cell subpopulations. Immunization of HVCN1-deficient mice with a T-dependent antigen resulted in a defect in CSR to all IgG subclasses, particularly marked for the IgG2b, whereas in contrast, no differences were observed in IgM secretion, suggesting a pivotal role for HVCN1 during antigen-driven B-cell activation and subsequent CSR. HVCN1 may influence B-cell activation through alteration of reactive oxygen species (ROS) as HVCN1-deficient B cells showed reduced ROS production following BCR activation, a sign of suboptimal NADPH oxidase activity. It has been postulated that proton channels are required to counterbalance the electrogenic activity of NADPH oxidase during ROS production. Our data suggest that this mechanism also occurs in vivo and shed new light on the role of ROS in B cell activation and downstream effects.

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