Role of histidines identified by mutagenesis in the NADPH oxidase-associated H+ channel.

The efflux of protons through a H+ channel acts as the charge compensation pathway for the electrogenic generation of superoxide (O-2) by human neutrophil NADPH oxidase. It has previously been shown that the N-terminal 230 amino acids of the product of the X-linked chronic granulomatous gene gp91(phox) contain all that is required for it to function as the arachidonate-activable, NADPH oxidase-associated H+ channel (Henderson, L. M., Thomas, S., Banting, G., and Chappell, J. B. (1997) Biochem. J. 325, 701-705). To identify functionally important amino acids, Chinese hamster ovary (CHO) cell lines were constructed that expressed point mutations in the N terminus of gp91(phox). No H+ flux was observed in CHO cell lines expressing the N-terminal gp91(phox) mutants H111L, H115L, and H119L, or H115L, or H115K. Partial retention of H+ channel function was, however, observed in the H115D CHO cell line. The addition of arachidonic acid to R91E,R92E CHO cells elicited a full H+ channel response. The buffering capacity and response of 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to H+ were the same in all cell lines. Therefore, it can be concluded that His-115 is important to the ability of gp91(phox) to function as the NADPH oxidase-associated H+ channel and that the mechanism of H+ conduction involves protonation and deprotonation of an amino acid with an appropriate pK value.