Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils

Generation of reactive oxygen species by the NADPH oxidase complex is an important bactericidal weapon of phagocytes, Phorbol myristate acetate (PMA) is a potent agonist for this "respiratory burst" in human neutrophils, Although stoichiometric H+ efflux occurs during the respiratory burst, efforts to stimulate voltage-gated H+ channels by PMA in whole-cell patch-clamped phagocytes have been unsuccessful. We have used a modification of the permeabilized-patch configuration that allows control of intracellular pH and preserves second-messenger pathways. Using this method, we show that PMA dramatically enhances and alters voltage-gated proton currents in human neutrophils. PMA produced four alterations in H+ current properties, each of which increases the H+ current at any given voltage: (i) a 40-mV negative shift in the H+ conductance-voltage (g(H)-V) relationship; (ii) faster activation [smaller activation time constant (tau(act))] during depolarizing pulses; (iii) slower deactivation [larger deactivation time constant (tau(tail))] on repolarization: and (iv) a larger maximum H+ conductance (g(H,max)). Inward current that directly reflects electron transport by NADPH oxidase was also activated by PMA stimulation. The identity of this electron current was confirmed by its sensitivity to diphenylene iodinium, an inhibitor of NADPH oxidase. Diphenylene iodinium also reversed the slowing of tau(tail) with a time course paralleling the inhibition of electron current. However, the amplitudes of H+ and electron currents activated by PMA were not correlated. A complex interaction between NADPH oxidase and voltage-gated proton channels is indicated. The data suggest that PMA stimulation modulates preexisting H+ channels rather than inducing a new H+ channel.