Identification and Characterization of Functional Residues in a Na+/H+ Antiporter (NhaA) from Escherichia coli by Random Mutagenesis

Forty-one mutants were isolated by means of random PCR mutagenesis of the Escherichia coli Na+/H+ antiporter (nhaA), which could not support the growth of a nhaAnhaB mutant (HIT ΔAB ) on plates containing 0.15 M LiCl (pH 7.5) or 0.65 M NaCl (pH 8.0). Most of the mutants were sensitive to both NaCl and LiCl, or to LiCl alone. DNA sequencing revealed that twelve of the mutants had single amino acid substitutions. All the mutations, except for H225P, were of the conserved residues of NhaA homologues and located in the putative transmembrane helices. The Na+/H+ and Li+/H+ antiporter activities of the mutant NhaA were measured with everted membrane vesicles: eight of the mutants lost both antiporter activities completely under all pH conditions examined. Although both D133A and L138P retained low Li+/H + antiporter activity, D133A lost Na+/H+ antiporter activity, while L138P retained normal Na+/H+ antiporter activity at pH 7.0 and 8.0. Interestingly, at pH 8.5, L138P no longer showed any Li+/ H+ antiporter activity. H225P retained relatively high antiporter activities, although their pH dependence was altered. These observations supported the previous indication that His-225 is part of the pH sensor [Gerchman, Y. et al. (1993) Proc. Natl. Acad. Set USA 90, 1212-1216]. L73R exhibited about 20% each of the activities only at pH 8.0, and showed a similar pH dependence to H225P in both Na+/H+ and Li+/H+ antiport. Therefore, in addition to His-225, Leu-73, and/or its vicinity may also contribute to the pH sensing.