Replacement of Lys-300 with a glutamine in the NhaA Na+/H+ antiporter of Escherichia coli yields a functional electrogenic transporter
| Autor: | Mehmet Karabel, Klaus Fendler, Miyer Patiño-Ruiz, Etana Padan, Octavian Călinescu, Manish Dwivedi |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
030102 biochemistry & molecular biology biology Stereochemistry Chemistry Antiporter Transporter Cell Biology Membrane transport Thermus thermophilus medicine.disease_cause biology.organism_classification Biochemistry 03 medical and health sciences 030104 developmental biology medicine Salt bridge Binding site Site-directed mutagenesis Molecular Biology Escherichia coli |
| Zdroj: | Journal of Biological Chemistry. 294:246-256 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|