Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase.

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.