Insight into the direct interaction of Na(+) with NhaA and mechanistic implications

Na(+)/H(+) antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life that are essential in cellular ion homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystals provided insight in the structure of this molecular machine. However, structural data revealing the composition of the binding site for Na(+) (or its surrogate Li(+)) is missing, representing a bottleneck in our understanding of the correlation between the structure and function of NhaA. Here, by adapting the scintillation proximity assay (SPA) for direct determination of Na(+) binding to NhaA, we revealed that (i) NhaA is well adapted as the main antiporter for Na(+) homeostasis in Escherichia coli and possibly in other bacteria as the cytoplasmic Na(+) concentration is similar to the Na(+) binding affinity of NhaA, (ii) experimental conditions affect NhaA-mediated cation binding, (iii) in addition to Na(+) and Li(+), the halide Tl(+) interacts with NhaA, (iv) whereas acidic pH inhibits maximum binding of Na(+) to NhaA, partial Na(+) binding by NhaA is independent of the pH, an important novel insight into the effect of pH on NhaA cation binding.