Voltage- and NADPH-dependence of electron currents generated by the phagocytic NADPH oxidase

The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O-2. The activity of the oxidase at the plasma membrane can be measured as electron current (I-e), and the voltage dependence of I-e was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from - 120 to 200 mV, were applied to obtain 'steady-state' current voltage relationships (I-V). The amplitude of I-e recorded at -40 mV was minimal at 8 mu M NADPH and saturated above I mM, with half-maximal activity (K-m) observed at approx. 110 mu M NADPH 0.1 mM NADPH, I-e was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (-60 to 60 mV), the I-V curve strongly rectifying only below - 100 mV. At lower NADPH concentrations the I-V curve was progressively shifted to more positive potentials and I-e became voltage-independent also within the physiological range. Consequently, the K of the oxidase decreased by approx. 40% (from 100 to 60 mu M) when the membrane potential increased from - 60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the I-e-V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of I-e reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not all intrinsic property of the oxidase.. Comparison of I-V values obtained at different NADPH concentrations revealed that the voltage-dependence of I-e is strongly influenced by the substrate concentration. Above