TY  - JOUR
A1  - Patiño-Ruiz, Miyer Fabián
A1  - Dwivedi, Manish
A1  - Călinescu, Octavian
A1  - Karabel, Mehmet
A1  - Padan, Etana
A1  - Fendler, Klaus
T1  - Replacement of Lys-300 with a glutamine in the NhaA Na+/H+ antiporter of Escherichia coli yields a functional electrogenic transporter
T2  - Journal of biological chemistry
N2  - Much of the research on Na+/H+ exchange has been done in prokaryotic models, mainly on the NhaA Na+/H+-exchanger from Escherichia coli (EcNhaA). Two conserved aspartate residues, Asp-163 and Asp-164, are essential for transport and are candidates for possible binding sites for the two H+ that are exchanged for one Na+ to make the overall transport process electrogenic. More recently, a proposed mechanism of transport for EcNhaA has suggested direct binding of one of the transported H+ to the conserved Lys-300 residue, a salt bridge partner of Asp-163. This contention is supported by a study reporting that substitution of the equivalent residue, Lys-305, of a related Na+/H+ antiporter, NapA from Thermus thermophilus, renders the transporter electroneutral. In this work, we sought to establish whether the Lys-300 residue and its partner Asp-163 are essential for the electrogenicity of EcNhaA. To that end, we replaced Lys-300 with Gln, either alone or together with the simultaneous substitution of Asp-163 with Asn, and characterized these transporter variants in electrophysiological experiments combined with H+ transport measurements and stability analysis. We found that K300Q EcNhaA can still support electrogenic Na+/H+ antiport in EcNhaA, but has reduced thermal stability. A parallel electrophysiological investigation of the K305Q variant of TtNapA revealed that it is also electrogenic. Furthermore, replacement of both salt bridge partners in the ion-binding site of EcNhaA produced an electrogenic variant (D163N/K300Q). Our findings indicate that alternative mechanisms sustain EcNhaA activity in the absence of canonical ion-binding residues and that the conserved lysines confer structural stability.
KW  - membrane transport
KW  - sodium-proton exchange
KW  - electrophysiology
KW  - fluorescence
KW  - site-directed mutagenesis
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/77598
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-775986
SN  - 0021-9258
VL  - 294
IS  - 1
SP  - 246
EP  - 256
PB  - American Society for Biochemistry and Molecular Biology Publications
CY  - Bethesda, Md
ER  -