Site-directed mutagenesis at the Glu78 in Ec-NhaA transporter impacting ion exchange: a biophysical study

Abstract

Na⁺/H⁺ antiporters facilitate the exchange of Na⁺ for H⁺ across the cytoplasmic membrane in prokaryotic and eukaryotic cells. These transporters are crucial to maintain the homeostasis of sodium ions, consequently pH, and volume of the cells. Therefore, sodium/proton antiporters are considered promising therapeutic targets in humans. The Na⁺/H⁺ antiporter in Escherichia coli (Ec-NhaA), a prototype of cation–proton antiporter (CPA) family, transports two protons and one sodium (or Li⁺) in opposite direction. Previous mutagenesis experiments on Ec-NhaA have proposed Asp164, Asp163, and Asp133 amino acids with the significant implication in functional and structural integrity and create site for ion-binding. However, the mechanism and the sites for the binding of the two protons remain unknown and controversial which could be critical for pH regulation. In this study, we have explored the role of Glu78 in the regulation of pH by Ec-NhaA. Although we have created various mutants, E78C has shown a considerable effect on the stoichiometry of NhaA and presented comparable phenotypes. The ITC experiment has shown the binding of ~ 5 protons in response to the transport of one lithium ion. The phenotype analysis on selective medium showed a significant expression compared to WT Ec-NhaA. This represents the importance of Glu78 in transporting the H⁺ across the membrane where a single mutation with Cys amino acid alters the number of H⁺ significantly maintaining the activity of the protein.