title: Mutation of Gly-94 in transmembrane segment M1 of Na+,K+-ATPase interferes with Na+ and K+ binding in E2P conformation.
abstract: The importance of Gly-93 and Gly-94 in transmembrane segment M1 of the Na+,K+-ATPase for interaction with Na+ and K+ was demonstrated by functional analysis of mutants Gly-93-Ala and Gly-94-Ala. In the crystal structures of the Ca2+-ATPase, the corresponding residues, Asp-59 and Leu-60, are located exactly where M1 bends. Rapid kinetic measurements of K+-induced dephosphorylation allowed determination of the affinity of the E2P phosphoenzyme intermediate for K+. In Gly-94-Ala, the K+ affinity was reduced 9-fold, i.e., to the same extent as seen for mutation of the cation-binding residue Glu-329. Furthermore, Gly-94-Ala showed strongly reduced sensitivity of the E1P-E2P equilibrium to Na+, with accumulation of E2P even at 600 mM Na+, indicating that interaction of E2P with extracellular Na+ is impaired. On the contrary, in Gly-93-Ala, the affinity for K+ was slightly increased, and the E1P-E2P equilibrium was displaced in favor of E1P. In both mutants, the affinity of the cytoplasmically facing sites of E1 for Na+ was reduced, but this effect was relatively small compared with the effects seen for E2P in Gly-94-Ala. Comparison with Ca2+-ATPase mutagenesis data suggests that the role of M1 in binding of the transported ions is universal among P-type ATPases, despite the low sequence homology in this region. Structural modeling of Na+,K+-ATPase mutant Gly-94-Ala on the basis of the Ca2+-ATPase crystal structures indicates that the alanine side chain comes close to Ile-287 of M3, particularly in E2P, thus resulting in a steric clash that may explain the present observations.
authors.affiliation: Department of Physiology, Institute of Physiology and Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Anja Pernille
authors.initials: AP
authors.lastname: Einholm
authors.forename: Mads
authors.initials: M
authors.lastname: Toustrup-Jensen
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
authors.forename: Bente
authors.initials: B
authors.lastname: Vilsen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010042:Ouabain; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium