Abstract
Na(+),K(+)-ATPase and H(+),K(+)-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H(+),K(+)-ATPase avoids binding of Na(+) at the site corresponding to the Na(+)-specific site of the Na(+),K(+)-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na(+),K(+)-ATPase with arginine, present in the H(+),K(+)-ATPase at the corresponding position, converted the normal 3Na(+):2K(+):1ATP stoichiometry of the Na(+),K(+)-ATPase to electroneutral 2Na(+):2K(+):1ATP stoichiometry similar to the electroneutral transport mode of the H(+),K(+)-ATPase. The electroneutral C932R mutant of the Na(+),K(+)-ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K(+) uptake. Only a relatively minor reduction of apparent Na(+) affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na(+) affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine(+) group of the M8 arginine replaces Na(+) at the third site, thus preventing Na(+) binding there, although allowing Na(+) to bind at the two other sites and become transported. Hence, in the H(+),K(+)-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.