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 (O(2)(-)) 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.
Morgan
Deri
D
Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, 1750 W Harrison, Chicago, IL 60612, USA.
Cherny
Vladimir V
VV
Price
Marianne O
MO
Dinauer
Mary C
MC
DeCoursey
Thomas E
TE
D000818:Animals; D019556:COS Cells; D002712:Chlorides / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism; D011522:Protons; D016897:Respiratory Burst / Q000502:physiology; D013481:Superoxides / Q000378:metabolism
D002712:Chlorides; D008562:Membrane Glycoproteins; D011522:Protons; D013481:Superoxides; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases