Analysis of cation specificity of NhaA, Na⁺/H⁺ antiporter by mutagenesis

Sodium/proton (Na⁺/H⁺) antiporters are membrane proteins that are important for sodium and pH homeostasis in cell and found almost in all eukaryotic and prokaryotic cells. These families of membrane proteins catalyse the exchange of H⁺/Na⁺ in cell and that are regulated by the variation of pH. In humans, they play vital role in the adjustment of cellular pH, cell volume, membrane potential and their dysfunction is associated with a variety of human diseases. No structure of eukaryotic antiporters has been determined and only succeeded to determine the structures of prokaryotic antiporters. Therefore, to understand ion binding site, topology and the position of key residues within the proteins, it is essential to study their kinetics and interactions. Our goal is to investigate the mechanism of ion selectivity in NhaA, which restricts it to the transport of only sodium or lithium ions in exchange for protons. Based on a structural motif, we built mutants in NhaA, on purpose to allow the protein to begin to transport potassium ions. We tested their growth phenotype and antiporter activity as compared to the WT. Here, we were able to interfere with the NhaA’s ability to transport sodium and lithium, implying that indeed the mutations most likely impaired the active site.