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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2017-01, Vol.114 (2), p.316-321
• Main Authors: Holm, Rikke, Khandelwal, Jaanki, Einholm, Anja P., Andersen, Jens P., Artigas, Pablo, Vilsen, Bente
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatase; Amino Acid Substitution; Arginine; Binding Sites; Binding, Competitive; Biological Sciences; Cations; Cell Membrane - enzymology; Cysteine; H(+)-K(+)-Exchanging ATPase - chemistry; H(+)-K(+)-Exchanging ATPase - genetics; Hemiplegia; Humans; Ion Channels; Ion Transport; Models, Molecular; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Phenylalanine; Phosphorylation; Potassium - metabolism; Protein Conformation; Protein Subunits - chemistry; Proton Pumps; Sequence Alignment; Sodium - metabolism; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1617951114

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.