Abstract:Na+/H+ antiporters in the CPA1 branch of the cation proton antiporter family drive the electroneutral exchange of H+ against Na+ ions and ensure pH homeostasis in eukaryotic and prokaryotic organisms. Although their transport cycle is overall electroneutral, specific partial reactions are electrogenic. Here, we present an electrophysiological study of the PaNhaP Na+/H+ antiporter from Pyrococcus abyssi reconstituted into liposomes. Positive transient currents were recorded upon addition of Na+ to PaNhaP proteo… Show more
“…jannaschii [ 22 ] and P . abyssi [ 39 ]. For these recordings, the transport currents were numerically reconstructed [ 40 ] and the stationary component of the reconstructed currents was determined ( S1 Fig ).…”
Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.
“…jannaschii [ 22 ] and P . abyssi [ 39 ]. For these recordings, the transport currents were numerically reconstructed [ 40 ] and the stationary component of the reconstructed currents was determined ( S1 Fig ).…”
Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.
“…Their physiological functions are also similar, that is, to take H + out of the cell in exchange for Na + . Pa NhaP has an even more acidic active pH range . Cryptococcus neoformans Nha1 is responsible for survival in acidic environments and the protein is active at pHs as low as 4.5 and Sp NHE1 is also active at more acidic extracellular pHs, with an optimum at approximately pH 4.5–5.0 .…”
Sodium proton antiporters (or sodium proton exchangers [NHEs]) are a critical family of membrane proteins that exchange sodium for protons across cell membranes. In yeast and plants, their primary function is to keep the sodium concentration low inside the cytoplasm. One class of NHE constitutively expressed in yeast is the plasma membrane Na 1 /H 1 antiporter, and another class is expressed on the endosomal/ vacuolar membrane. At present, four bacterial plasma membrane antiporter structures are known and nuclear magnetic resonance structures are available for the membrane spanning transmembrane helices of mammalian and yeast NHEs.Additionally, a vast amount of mutational data are available on the role of individual amino acids and critical motifs involved in transport. We combine this information to obtain a more detailed picture of the yeast NHE plasma membrane protein and review mechanisms of transport, conserved motifs, unique residues important in function, and regulation of these proteins. The Na
“…3a) was markedly different to both the WT and the previously described mutants. Thus, the transient currents recorded at pH 9 and 9.5 for a Na + concentration jump of 100 mM have a major, positive component, that has a fast decay characteristic of a pre-steady-state charge displacement 35 .Figure 3Electrophysiological characterization of KpNhaB D146A. ( a ) Transient currents recorded for KpNhaB D146A following Na + concentration jumps of 100 mM under different pH conditions.…”
Section: Resultsmentioning
confidence: 99%
“…While there seems to be no a priori requirement to have a negatively charged secondary chain in order for an amino acid residue to coordinate sodium in a typical binding site 38 , a special consideration has to be given for the binding sites of Na + /H + exchangers. Experimental evidence collected on several prokaryotic Na + /H + exchangers, including KpNhaB, shows that the transport mechanism of Na + /H + exchangers is explained by competition of Na + and H + for a common binding site 26,31,35,36 . As such, a common binding site for H + and Na + requires the existence of at least one protonatable residue.…”
Bacterial NhaB Na+/H+ exchangers belonging to the Ion Transporter superfamily are poorly characterized in contrast to Na+/H+ exchangers of the Cation Proton Antiporter superfamily which have NhaA from Escherichia coli as a prominent member. For a more detailed understanding of the intricacies of the exchanger’s transport mechanism, mutational studies are essential. Therefore, we mutated two protonatable residues present in the putative transmembrane region of NhaB from Klebsiella pneumoniae (KpNhaB), which could serve as substrate binding sites, Asp146 and Asp404, to either glutamate or alanine and analyzed transport function and stability of the mutants using electrophysiological and fluorimetric techniques. While mutation of either Asp residue to Glu only had slight to moderate effects on the transport activity of the exchanger, the mutations D404A and D146A, in particular, had more profound effects on the transport function. Furthermore, a double mutant, D146A/D404A, exhibited a remarkable behavior at alkaline pH, where recorded electrical currents changed polarity, showing steady-state transport with a stoichiometry of H+:Na+ < 1, as opposed to the H+:Na+ > 1 stoichiometry of the WT. Thus, we showed that Asp146 and Asp404 are part of the substrate binding site(s) of KpNhaB and engineered a Na+/H+ exchanger with a variable stoichiometry.
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