THE Ec-NhaA TRANSPORTER SWITCHES FROM ANTAGONISTIC TO SYNERGISTIC ANTIPORTING UPON A SINGLE POINT MUTATION

Living cells are critically dependent on homeostasis of pH, Na⁺ and volume in which Na⁺/H⁺ antiporters are involved. The Na⁺/H⁺ antiporter (Ec-NhaA) of Escherichia coli exchanges Na⁺ or Li⁺ for H⁺. We used isothermal titrations calorimetry to perform a detailed stoichiometric and thermodynamic analysis of Li⁺ binding to Ec-NhaA and several of its mutants. We found that, in-line with the canonical alternative access mechanistic model of secondary transporters, Li⁺/H⁺ binding to the antiporter is antagonistically coupled. Binding of Li⁺ displaces 2 H⁺ from the binding site. Mutations in the Li⁺ binding site (D163E, D163N, D164N, D164E) all affected Li⁺ binding, H⁺ release and antiporter activity to the same extent, while D133C changed the H⁺/Li⁺ stoichiometry to 4. Most striking, however, was the single point mutation near the active site, A167P, which converted the Ec-NhaA antagonistic binding to a synergistic binding of 6H⁺/Li⁺. Synergistic binding is known to occur in certain anionic transporters, such as Ec1-CLC, the Cl^-/H⁺ antiporter.