Replacements of histidine 226 of NhaA-Na+/H+ antiporter of Escherichia coli. Cysteine (H226C) or serine (H226S) retain both normal activity and pH sensitivity, aspartate (H226D) shifts the pH profile toward basic pH, and alanine (H226A) inactivates the carrier at all pH values.

We have previously shown that replacement of His-226 in the NhaA Na+/H+ antiporter of Escherichia coli to Arg (H226R) shifts the pH profile of the antiporter toward acidic pH and as a result of delta nhaA delta nhaB strain bearing this mutation is Na+ sensitive at alkaline pH (Gerchman, Y., Olami, Y., Rimon, A., Taglicht, D., Schuldiner, S. and Padan, E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1212-1216). In the present work the role of His-226 in the response of NhaA to pH has been studied in detail. The Na+ sensitivity of the delta nhaA delta nhaB mutant bearing the H226R-NhaA plasmid at alkaline pH provided a very powerful tool to isolate revertants and suppressants of H226R growing on high Na+ at alkaline pH. With this approach cysteine (H226C) and serine (H226S) replacements were found to efficiently replace His-226 and yield an antiporter, which like the wild-type protein, is activated by pH between pH 7 and 8. These results imply that polarity and/or hydrogen bonding, the common properties shared by these amino acid residues, are essential at position 226 for pH regulation of NhaA. This suggestion was substantiated by site-directed mutagenesis of His-226 either to alanine (H226A) or aspartate (H226D). Whereas H226A-NhaA shows very low activity which is not activated by pH, H226D-NhaA is active and regulated by pH. The pH profile of H226D is shifted by half a pH unit toward alkaline pH, as opposed to the previously isolated mutant H226R which has a pH profile shift, to the same extent, but toward acidic pH. It is suggested that charge modifies the pH profile but is not essential for the pH regulation of NhaA.