Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O₂^-) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H⁺ efflux was thought to be contained within the gp91^phox subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS_phox). The 7D5 antibody, which detects an extracellular epitope of the gp91^phox protein, labeled 96-98% of COS_phox cells. NADPH oxidase was functional because COS_phox (but not COS_WT) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS_WT) or COS_phox cells studied at pH_o 7.0 and pH_i 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H⁺ current in COS_WT or COS_phox cells. Therefore, gp91^phox does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.