Expression of gp91^phox/Nox2 in COS-7 cells: cellular localization of the protein and the detection of outward proton currents

We have reported previously that gp91^phox, expressed in CHO (Chinese hamster ovary) cells, functions as a voltage-dependent proton channel. However, others have reported that COS-7 cells expressing gp91^phox failed to exhibit outward proton currents, and concluded that gp91^phox does not function as a proton channel. To investigate this clear difference in findings, we have examined the expression and cellular localization of the fusion protein EGFP-C–91, in which gp91^phox is fused to the C-terminus of enhanced green fluorescent protein. EGFP-C–91 was observed in the plasma membrane and intracellular membranes of 30% of the transfected COS-7 cells. In the remaining COS-7 cells, EGFP-C–91 was detected in the intracellular membranes only. In CHO cells EGFP-C–91 was present in both the plasma membrane and the intracellular membranes of all transfected cells. Under the whole-cell configuration, outward currents were recorded from COS-7 cells expressing gp91^phox. These increased in magnitude and lost their ‘droop’ over time as the pipette solution equilibrated with the cell cytoplasm (50 min). The threshold activation voltage for the currents was shifted by ∼60 mV for a 1 unit difference in bath pH. Zn²⁺ inhibited the outward currents observed in COS-7 cells expressing gp91^phox. The tail current reversal potential was −64 mV at a pH_o (external pH) of 8.0, −40 mV at pH_o 7.4 and −8 mV at pH_o 7.0, indicating that the current arises from the movement of protons. Outward currents were exhibited by 37.5% of the COS-7 cells expressing gp91^phox. Proton currents were recorded following the excision of inside-out patches from cells transfected with gp91^phox. The presence of outward proton currents in COS-7 cells expressing gp91^phox provides further support for our proposed role for gp91^phox as the NADPH oxidase-associated proton channel.