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

Abstract

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,KATPase 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 thepHis 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 pKa 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.