Transmembrane segment II of NhaA Na+/H+ antiporter lines the cation passage, and Asp65 is critical for pH activation of the antiporter.

The Journal of Biological Chemistry
K HerzE Padan

Abstract

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 5.5-8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent K(m) to Na+ and Li+ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [14C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents,...Continue Reading

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Citations

Sep 26, 2013·Nature Communications·Kalyan C KondapalliRajini Rao
Apr 6, 2011·Nature Reviews. Microbiology·Terry A KrulwichEtana Padan
Aug 30, 2011·Proceedings of the National Academy of Sciences of the United States of America·Lena Kozachkov, Etana Padan
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Feb 10, 2021·Nature Plants·Huei-Hsuan Tsai, Wolfgang Schmidt
Dec 12, 2012·Journal of Molecular and Cellular Cardiology·Brian L LeeLarry Fliegel

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