The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (I(e)) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that I(e) is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates I(e), because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage gated proton channel1, hHV1. This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes2–4, histamine secretion by basophils5, sperm capacitation6, and airway pH7. The most selective ion channel known, HV1 shows no detectable permeability to other ions1. Opposing classes of selectivity mechanisms postulate that (a) a titratable amino acid residue in the permeation pathway imparts proton selectivity1, 8–11, or (b) water molecules “frozen” in a narrow pore conduct protons while excluding other ions12. Here we identify Aspartate112 as a crucial component of the selectivity filter of hHV1. When a neutral amino acid replaced Asp112, the mutant channel lost proton specificity and became anion selective or did not conduct. Only the glutamate mutant remained proton specific. Mutation of the nearby Asp185 did not impair proton selectivity, suggesting that Asp112 plays a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp112, the mutant channel was still anion permeable. Evidently, the proton specificity of hHV1 requires an acidic group at the selectivity filter.

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.

Fogel and Hastings first hypothesized the existence of voltagegated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kH V 1: it is proton-specific and activated by depolarization, its g H -V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp 51 ) results in loss of proton-specific conduction. Indirect evidence suggests that kH V 1 is monomeric, unlike other proton channels. Furthermore, kH V 1 differs from all known proton channels in activating well negative to the Nernst potential for protons, E H . This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.ion selectivity | ion channel | permeation | channel gating | action potential