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

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.