A universal mechanism for transport and regulation of CPA sodium proton exchangers

Călinescu, Octavian; Fendler, Klaus

doi:10.1515/hsz-2014-0278

Cited by 17 publications

(31 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A bell-shaped pH dependence ( Fig. 5c) and increment in K m Naϩ values at acidic pH (Table 2) suggest a competition-based transport mechanism of TtNapA similar to that of other CPA2 Na ϩ /H ϩ antiporters (30). Optimal pH and Na ϩ affinity are similar to WT EcNhaA (Tables 1 and 2).…”

Section: K300q Nhaa Is Functional and Electrogenicmentioning

confidence: 60%

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

Patiño-Ruiz

Dwivedi

Călinescu

et al. 2019

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: K300q Nhaa Is Functional and Electrogenicmentioning

confidence: 60%

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

Patiño-Ruiz

Dwivedi

Călinescu

et al. 2019

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Unlike electroneutral CPA1 antiporters, microbial NhaA transporters of the CPA2 subtype mediate electrogenic exchange of 2H + for Na + , allowing them to extrude Na + against an inwardly directed electrochemical gradient. Consistent with a physiological function in Na + secretion, NhaA members exhibit alkaline pKa for transport, which favors periplasmic H + binding [2]. Similar to NhaA, human NHA2 appears more active at neutral to alkaline pH, although evidence is still lacking for electrogenic transport [14].…”

Section: Resultsmentioning

confidence: 99%

NHA2 is expressed in distal nephron and regulated by dietary sodium

et al. 2016

View full text Add to dashboard Cite

Increased renal reabsorption of sodium is a significant risk factor in hypertension. An established clinical marker for essential hypertension is elevated sodium lithium countertransport (SLC) activity. NHA2 is a newly identified Na+(Li+)/H+ antiporter with potential genetic links to hypertension, which has been shown to mediate SLC activity and H+-coupled Na+(Li+) efflux in kidney-derived MDCK cells. To evaluate a putative role in sodium homeostasis, we determined the effect of dietary salt on NHA2. In murine kidney sections, NHA2 localized apically to distal convoluted (both DCT1 and 2) and connecting tubules, partially overlapping in distribution with V-ATPase, AQP2, and NCC1 transporters. Mice fed a diet high in sodium chloride showed elevated transcripts and expression of NHA2 protein. We propose a model in which NHA2 plays a dual role in salt reabsorption or secretion, depending on the coupling ion (sodium or protons). The identified novel regulation of Na+/H+ antiporter in the kidney suggests new roles in salt homeostasis and disease.

show abstract

“…An effective strictly coupled Na + /H + exchange process requires that only the fully loaded transporter (2 H + or 1 Na + bound in the case of NhaA) can perform this transition and that it is inhibited in the empty apo transporter (46). We suggest that the formation of the Lys-300–Asp-163 salt bridge may be an inhibitory element by making the structure more rigid so that the energetic barrier for the conformational transition of the apo transporter is increased.…”

Section: Discussionmentioning

confidence: 99%

Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na+/H+ antiporter

Călinescu

Dwivedi

Patiño-Ruiz

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Na+/H+ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na+, and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na+/H+ exchanger family and a model system for all related Na+/H+ exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H+ ions that NhaA exchanges for one Na+ ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na+ and H+ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na+/H+ exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300.

show abstract

A universal mechanism for transport and regulation of CPA sodium proton exchangers

Cited by 17 publications

References 19 publications

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

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

NHA2 is expressed in distal nephron and regulated by dietary sodium

Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na+/H+ antiporter

Contact Info

Product

Resources

About