title: 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.
authors.affiliation: Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Holm
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430.
authors.forename: Jaanki
authors.initials: J
authors.lastname: Khandelwal
authors.affiliation: Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Anja P
authors.initials: AP
authors.lastname: Einholm
authors.affiliation: Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Jens P
authors.initials: JP
authors.lastname: Andersen
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430 bv@biomed.au.dk pablo.artigas@ttuhsc.edu.
authors.forename: Pablo
authors.initials: P
authors.lastname: Artigas
authors.affiliation: Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark; bv@biomed.au.dk pablo.artigas@ttuhsc.edu.
authors.forename: Bente
authors.initials: B
authors.lastname: Vilsen
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine; D001665:Binding Sites; D001667:Binding, Competitive; D002412:Cations; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics; D006429:Hemiplegia; D006801:Humans; D007473:Ion Channels; D017136:Ion Transport; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D017494:Proton Pumps; D016415:Sequence Alignment; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics
chemical_list: D002412:Cations; D007473:Ion Channels; D021122:Protein Subunits; D017494:Proton Pumps; D010649:Phenylalanine; D001120:Arginine; D012964:Sodium; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine; D011188:Potassium
keywords: H+,K+-pump; Na+,K+-pump; alternating hemiplegia of childhood; electrogenicity; internal cation