Voltage-activated H + currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, V,~, was close to the Nernst potential, Em varying 52 mV/U pH over four ApH units (ApH = pHo-pHi). This result indicates that H + channels are extremely selective, PH/PT~ > 107, and that both internal and external pH, pHi, and pHo, were well controlled. The H + current amplitude was practically Constant at any fixed ApH, in spite of up to 100-fold symmetrical changes in H § concentration. Thus, the rate-limiting step in H + permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current-voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H + channels pass through at least two closed states before opening. The steady state H + conductance, ga, as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by ~ 40 mV/U pH by changes in pHi or pHo, with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H + currents were detectably activated can be described empirically as ~ 20--40(pHo-pHi) mV. If internal and external protons regulate the voltage dependence of gH gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, ApH. We propose a simple general model to account for the observed ApH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within Address correspondence to Thomas E.

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.