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 Delta AB(-)) on plates containin g 0.15 M LiCl (pH 7.5) or 0.65 M NaCl (pH 8.0). Most of the mutants we re sensitive to both NaCl and LiCl, or to LiCl alone, DNA sequencing r evealed 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 me asured with everted membrane vesicles: eight of the mutants lost both antiporter activities completely under all pH conditions examined, Alt hough both D133A and L138P retained low Li+/H+ antiporter activity, D1 33A 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, L13 8P no longer showed any Li+/H+ antiporter activity, H225P retained rel atively high antiporter activities, although their pH dependence was a ltered, These observations supported the previous indication that His- 225 is part of the pH sensor [Gerchman, Y. et al. (1993) Proc, Natl, A cad, Sci, USA 90, 1212-1216], L73R exhibited about 20% each of the act ivities 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, L eu-73, and/or its vicinity may also contribute to the pH sensing.