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Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter

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Olkhova,  Elena
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Herz, K., Rimon, A., Olkhova, E., Kozachkov, L., & Padan, E. (2010). 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, 285(3), 2211-2220.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-D716-8
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 Km 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, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent Km of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp65 negative charge for pH activation of the antiporter.