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Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: An electrophysiological study

MPG-Autoren
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Mager,  Thomas
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Fendler,  Klaus
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Zitation

Mager, T., Rimon, A., Padan, E., & Fendler, K. (2011). Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: An electrophysiological study. Journal of Biological Chemistry, 286(26), 23570-23581.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0024-D5E2-A
Zusammenfassung
Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na+ or H+ gradients as the driving force. Under symmetrical pH conditions with a Na+ gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the Km for Na+ at acidic pH. Under symmetrical Na+ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na+ and H+ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na+ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na+ translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.