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.
Na+,K+-ATPase actively extrudes three cytoplasmic Na+ ions in exchange for two extracellular K+ ions for each ATP hydrolyzed. The atomic structure with bound Na+ identifies three Na+ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na+ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na+ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na+ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na+ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na+ affinity. This indicates that Na+ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E1-like configuration, allowing Na+ but not K+ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.
more » « less- Award ID(s):
- 2003251
- PAR ID:
- 10499699
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Biomolecules
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2218-273X
- Page Range / eLocation ID:
- 135
- Subject(s) / Keyword(s):
- Na+,K+-pump Na+ site K+ site Na+ affinity K+ affinity P-type ATPase mutagenesis serine phosphorylation electrophysiology
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/biom14010135
