3 years ago

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

Miyer Patiño-Ruiz, Manish Dwivedi, Octavian Călinescu, Mehmet Karabel, Etana Padan, Klaus Fendler
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.

Publisher URL: http://doi.org/10.1074/jbc.RA118.004903

DOI: 10.1074/jbc.RA118.004903

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