Selectivity of externally facing ion-binding sites in the Na/K pump to alkali metals and organic cations

The Na/K pump is a P-type ATPase that exchanges three intracel- lular Na+ ions for two extracellular K+ ions through the plasma- lemma of nearly all animal cells. The mechanisms involved in cation selection by the pump’s ion-binding sites (site I and site II bind either Na+ or K+; site III binds only Na+) are poorly understood. We studied cation selectivity by outward-facing sites (high K+ af- finity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium+, methylguanidinium+, and aminoguanidi- nium+ produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly di- rected pump current at saturating K+, and (ii) induction of pump- mediated, guanidinium-derivative–carried inward current at nega- tive potentials without Na+ and K+. In contrast, formamidinium+ and acetamidinium+ induced K+-like outward currents. Measure- ment of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K+ congeners. Molecular dynamics simulations indicate that bound organic cati- ons induce minor distortion of the binding sites. Among tested metals, only Li+ induced Na+-like VDI, whereas all metals tested except Na+ induced K+-like outward currents. Pump-mediated K+- like organic cation transport challenges the concept of rigid struc- tural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Further- more, actions by guanidinium+ derivatives suggest that Na+ binds to site III in a hydrated form and that the inward current observed without external Na+ and K+ represents cation transport when nor- mal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites.