Three aspartic residues in membrane-spanning regions of Na +/H + antiporter from Vibrio alginolyticus play a role in the activity of the carrier

Abstract

The Na +/H + antiporter gene from Vibrio alginolyticus restores the growth of an nhaA-defective strain of Escherichia coli, NM81, in a high NaCl medium (Nakamura, T., Komano, Y., Itaya, E., Tsukamoto, K., Tsuchiya, T. and Unemoto, T. (1994) Biochim. Biophys. Acta 1190, 465–468). This gene, named nhaAv, allowed the nhaA-defective E. coli strains, NM81( ΔnhaA) and RS1 ( ΔnhaA, chaA −), to extrude Na + at alkaline pH. The extrusion of Na + occurred against its chemical gradient in the presence of membrane-permeable amine. Thus, the nhaAv gene product is functional as an electrogenic Na +/H + antiporter in E. coli cells. The NhaAv protein has only four acidic amino acid residues in the putative membrane-spanning regions, that is, Asp-57, Asp-125, Asp-155 and Asp-156, and these Asp residues are conserved in NhaA from E. coli. Asp-111, which is predicted to be in a loop region between the transmembrane segments is also conserved in NhaA. Thus, each conserved Asp residue was replaced with asparagine by a site-directed mutagenesis. E. coli NM81 cells containing a plasmid harboring the nhaAv gene mutated at Asp-125, −155 or −156 could neither grow in a high NaCl medium nor extrude Na + at alkaline pH against its chemical gradient. These results show that Asp-125, −155 and −156, but not Asp-57 and −111, play a role in the activity of the Na +/H + antiporter, NhaAv.