TY  - JOUR
A1  - Călinescu, Octavian
A1  - Dwivedi, Manish
A1  - Patiño-Ruiz, Miyer Fabián
A1  - Padan, Etana
A1  - Fendler, Klaus
T1  - Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na+/H+ antiporter
T2  - The journal of biological chemistry
N2  - 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.
KW  - electrophysiology
KW  - enzyme mechanism
KW  - membrane transport
KW  - secondary active transport
KW  - site-directed mutagenesis
KW  - sodium-proton exchange
KW  - transport mechanism
KW  - transporter
Y1  - 2017
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/45818
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-458181
SN  - 0021-9258
SN  - 1083-351X
N1  - Free via Creative Commons: CC-BY license
VL  - 292
IS  - 19
SP  - 7932
EP  - 7941
PB  - ASBMB
CY  - Bethesda, Md.
ER  -