Three aspartic residues in membrane-spanning regions of Na+/H+ antiporter from Vibrio alginolyticus play a role in the activity of the carrier.
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(delta nhaA) and RS1 (delta nhaA, 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.
Nakamura
T
T
Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Komano
Y
Y
Unemoto
T
T
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D012966:Sodium Cyanide / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014733:Vibrio / Q000235:genetics / Q000378:metabolism*
D011522:Protons; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D001224:Aspartic Acid; D000584:Amiloride; D012964:Sodium; D012966:Sodium Cyanide; C039614:ethylisopropylamiloride