pThe phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular Osub2/sub. The activity of the oxidase at the plasma membrane can be measured as electron current (iI/isube/sub), and the voltage dependence of iI/isube/sub 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 iI/isube/sub recorded at ?40 mV was minimal at 8 μM NADPH and saturated above 1 mM, with half-maximal activity (iK/isubm/sub) observed at approx. 110 μM NADPH. Comparison of I–V values obtained at different NADPH concentrations revealed that the voltage-dependence of iI/isube/sub is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, iI/isube/sub 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 iI/isube/sub became voltage-independent also within the physiological range. Consequently, the iK/isubm/sub of the oxidase decreased by approx. 40% (from 100 to 60 μM) 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 Isube/sub–V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of iI/isube/sub reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase./p
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机译：>吞噬NADPH氧化酶通过将电子从胞质NADPH转移到细胞外O 2 来产生超氧化物。质膜上氧化酶的活性可以用电子电流（ I e ）和 I 的电压依赖性来测量据报道，e 在人类嗜酸性粒细胞中表现出强整流作用，电流在负电位下几乎与电压无关。为了研究潜在的机制，我们对由PMA激活的人嗜酸性粒细胞的由内而外的贴片进行了电压钳实验。通过浴液施加不同浓度的NADPH诱发电子电流，而施加从120到200mV的缓慢电压斜坡（0.8nV / ms），以获得“稳态”电流-电压关系（I –V）。在<40> mV处记录的 I e 的振幅在8μMNADPH时最小，在1mM以上饱和，具有最大活性的一半（ K < / i> m ）在NADPH约为110nM。比较在不同NADPH浓度下获得的I–V值，发现 I e 的电压依赖性受底物浓度的强烈影响。高于0.1mM NADPH时， I e 显着依赖电压，并且在生理膜电位范围（？60至60mV）内随着去极化而急剧下降。 -V曲线仅在？100 mV以下才有力地整流。在较低的NADPH浓度下，IV曲线逐渐移向更高的正电位，并且在生理范围内， i e 也变得与电压无关。因此，当膜电位从？60升高到60mV时，氧化酶的 K m 降低了约40％（从100降低到60μM）。 。我们得出的结论是，氧化酶的活性取决于膜电位和[NADPH]，并且I e -V曲线的形状受亚毫摩尔范围内NADPH浓度的影响。全细胞穿孔贴片记录中报道的 I e 令人惊讶的电压独立性很可能是由于底物限制所致，而不是氧化酶的固有特性。
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