Replacement of Lys-300 with a glutamine in the NhaA Na+/H+ antiporter of Escherichia coli yields a functional electrogenic transporter
- 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.
Verfasserangaben: | Miyer Fabián Patiño-RuizORCiDGND, Manish DwivediORCiD, Octavian CălinescuORCiDGND, Mehmet Karabel, Etana PadanORCiD, Klaus FendlerORCiDGND |
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URN: | urn:nbn:de:hebis:30:3-775986 |
DOI: | https://doi.org/10.1074/jbc.RA118.004903 |
ISSN: | 0021-9258 |
Pubmed-Id: | https://pubmed.ncbi.nlm.nih.gov/30409911 |
Titel des übergeordneten Werkes (Englisch): | Journal of biological chemistry |
Verlag: | American Society for Biochemistry and Molecular Biology Publications |
Verlagsort: | Bethesda, Md |
Dokumentart: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Datum der Veröffentlichung (online): | 04.01.2021 |
Jahr der Erstveröffentlichung: | 2019 |
Veröffentlichende Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Datum der Freischaltung: | 03.02.2024 |
Freies Schlagwort / Tag: | electrophysiology; fluorescence; membrane transport; site-directed mutagenesis; sodium-proton exchange |
Jahrgang: | 294 |
Ausgabe / Heft: | 1 |
Seitenzahl: | 11 |
Erste Seite: | 246 |
Letzte Seite: | 256 |
Institute: | Biochemie, Chemie und Pharmazie / Biochemie und Chemie |
Angeschlossene und kooperierende Institutionen / MPI für Biophysik | |
DDC-Klassifikation: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Lizenz (Deutsch): | Creative Commons - CC BY - Namensnennung 4.0 International |