Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na+/H+ antiporter

  • Na+/H+ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na+, and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na+/H+ exchanger family and a model system for all related Na+/H+ exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H+ ions that NhaA exchanges for one Na+ ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na+ and H+ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na+/H+ exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300.

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Metadaten
Author:Octavian Călinescu, Manish Dwivedi, Miyer Patino-Ruiz, Etana Padan, Klaus Fendler
URN:urn:nbn:de:hebis:30:3-458181
DOI:https://doi.org/10.1074/jbc.M117.778175
ISSN:0021-9258
ISSN:1083-351X
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/28330875
Parent Title (English):The journal of biological chemistry
Publisher:ASBMB
Place of publication:Bethesda, Md.
Contributor(s):Thomas Söllner
Document Type:Article
Language:English
Year of Completion:2017
Date of first Publication:2017/03/22
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2018/03/05
Tag:electrophysiology; enzyme mechanism; membrane transport; secondary active transport; site-directed mutagenesis; sodium-proton exchange; transport mechanism; transporter
Volume:292
Issue:19
Page Number:11
First Page:7932
Last Page:7941
Note:
Free via Creative Commons: CC-BY license
HeBIS-PPN:432102140
Institutes:Biochemie, Chemie und Pharmazie / Pharmazie
Angeschlossene und kooperierende Institutionen / MPI für Biophysik
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Sammlungen:Universitätspublikationen
Licence (German):License LogoCreative Commons - Namensnennung 4.0