Lysine 300 is essential for stability but not for electrogenic transport of theEscherichia coliNhaA 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 ofEscherichia coliis 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.
From the Max-Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.|the "Carol Davila" University of Medicine and Pharmacy, 050474 Bucharest, Romania, and.
Octavian
O
Călinescu
the Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Manish
M
Dwivedi
From the Max-Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.
Miyer
M
Patiño-Ruiz
the Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Etana
E
Padan
From the Max-Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany, fendler@biophys.mpg.de.
Klaus
K
Fendler
D001692:Biological Transport; D001693:Biological Transport, Active; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005470:Fluorometry; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D017433:Protein Structure, Secondary; D021381:Protein Transport; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D008239:Lysine
electrophysiology; enzyme mechanism; membrane transport; secondary active transport; site-directed mutagenesis; sodium-proton exchange; transport mechanism; transporter