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  Replacement of Lys-300 with a glutamine in the NhaA Na+/H+ antiporter of Escherichia coli yields a functional electrogenic transporter

Patiño-Ruiz, M., Dwivedi, M., Călinescu, O., Karabel, M., Padan, E., & Fendler, K. (2019). Replacement of Lys-300 with a glutamine in the NhaA Na+/H+ antiporter of Escherichia coli yields a functional electrogenic transporter. The Journal of Biological Chemistry, 294(1), 246-256. doi:10.1074/jbc.RA118.004903.

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 Creators:
Patiño-Ruiz, Miyer1, Author           
Dwivedi, Manish2, Author
Călinescu, Octavian1, 3, Author           
Karabel, Mehmet1, Author           
Padan, Etana2, Author
Fendler, Klaus1, Author           
Affiliations:
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              
2Institute of Life Sciences, Hebrew University of Jerusalem, Israel, ou_persistent22              
3Department of Biophysics, "Carol Davila" University of Medicine and Pharmacy, 050474 Bucharest, Romania, ou_persistent22              

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Free keywords: electrophysiology; fluorescence; membrane transport; site-directed mutagenesis; sodium-proton exchange
 Abstract: 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 relatedNa+/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.

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Language(s): eng - English
 Dates: 2018-07-182018-11-062018-11-082019-01-04
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1074/jbc.RA118.004903
 Degree: -

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Title: The Journal of Biological Chemistry
  Other : JBC
Source Genre: Journal
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Publ. Info: Baltimore, etc. : American Society for Biochemistry and Molecular Biology [etc.]
Pages: - Volume / Issue: 294 (1) Sequence Number: - Start / End Page: 246 - 256 Identifier: ISSN: 0021-9258
CoNE: https://pure.mpg.de/cone/journals/resource/954925410826_1