Distinct pH dependencies of Na⁺/K⁺ selectivity at the two faces of Na,K-ATPase

The sodium pump (Na,K-ATPase) in animal cells is vital for actively maintaining ATP hydrolysis-powered Na⁺ and K⁺ electrochemical gradients across the cell membrane. These ion gradients drive co- and countertransport and are critical for establishing the membrane potential. It has been an enigma how Na,K-ATPase discriminates between Na⁺ and K⁺, despite the pumped ion on each side being at a lower concentration than the other ion. Recent crystal structures of analogs of the intermediate conformations E2·Pi·2K⁺ and Na⁺-bound E1∼P·ADP suggest that the dimensions of the respective binding sites in Na,K-ATPase are crucial in determining its selectivity. Here, we found that the selectivity at each membrane face is pH-dependent and that this dependence is unique for each face. Most notable was a strong increase in the specific affinity for K⁺ at the extracellular face (i.e. E2 conformation) as the pH is lowered from 7.5 to 5. We also observed a smaller increase in affinity for K⁺ on the cytoplasmic side (E1 conformation), which reduced the selectivity for Na⁺ Theoretical analysis of the pK values of ion-coordinating acidic amino acid residues suggested that the face-specific pH dependences and Na⁺/K⁺ selectivities may arise from the protonation or ionization of key residues. The increase in K⁺ selectivity at low pH on the cytoplasmic face, for instance, appeared to be associated with Asp⁸⁰⁸ protonation. We conclude that changes in the ionization state of coordinating residues in Na,K-ATPase could contribute to altering face-specific ion selectivity.