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 ...