Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase

• Published in: Scientific reports 2019-09, Vol.9 (1), p.13344-14, Article 13344
• Main Authors: Nielsen, Hang N., Spontarelli, Kerri, Holm, Rikke, Andersen, Jens Peter, Einholm, Anja P., Artigas, Pablo, Vilsen, Bente
• Format: Article
• Language: English
• Subjects: 13; 13/106; 13/109; 38/70; 631/45/173; 631/57/2283; 82; 82/80; Amino Acid Substitution - genetics; Animals; Binding sites; Binding Sites - genetics; Cell Line; Cell Membrane - metabolism; Cell Proliferation - genetics; Chlorocebus aethiops; COS Cells; Crystal structure; Gating; Glutamine; Humanities and Social Sciences; Intracellular; Models, Molecular; multidisciplinary; Mutagenesis, Site-Directed; Mutation; Na+/K+-exchanging ATPase; Phosphorylation - genetics; Potassium - metabolism; Protein Binding - physiology; Protein Conformation; Science; Science (multidisciplinary); Sodium - metabolism; Sodium-Potassium-Exchanging ATPase - genetics; Sodium-Potassium-Exchanging ATPase - metabolism; Translocation; Xenopus
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-50009-2

Three Na + sites are defined in the Na + -bound crystal structure of Na + , K + -ATPase. Sites I and II overlap with two K + sites in the K + -bound structure, whereas site III is unique and Na + specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na + at site III, but does not contribute to K + coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na + , K + -ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na + and K + binding properties. Remarkably, Q925 substitutions had minor effects on Na + binding from the intracellular side of the membrane – in fact, mutations Q925A and Q925G increased the apparent Na + affinity – but caused dramatic reductions of the binding of K + as well as Na + from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na + -binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na + at the extracellular side.