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.
Henderson
L M
LM
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. L.M.Henderson@bris.ac.uk
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*
D017931:DNA Primers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007473:Ion Channels; D008562:Membrane Glycoproteins; D016718:Arachidonic Acid; D006639:Histidine; D006859:Hydrogen; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases