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 proteoliposomes, indicating a reaction where positive charge is rapidly displaced into the proteoliposomes with a rate constant of k >200 s−1. We attribute the recorded currents to an electrogenic reaction that includes Na+ binding and possibly occlusion. Subsequently, positive charge is transported out of the cell associated with H+ binding, so that the overall reaction is electroneutral. We show that the differences in pH profile and Na+ affinity of PaNhaP and the related MjNhaP1 from Methanocaldococcus jannaschii can be attributed to an additional negatively charged glutamate residue in PaNhaP. The results are discussed in the context of the physiological function of PaNhaP and other microbial Na+/H+ exchangers. We propose that both, electroneutral and electrogenic Na+/H+ antiporters, represent a carefully tuned self-regulatory system, which drives the cytoplasmic pH back to neutral after any deviation.
Highlights
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
In a recent study we demonstrated that the competitionbased transport mechanism first proposed for the electrogenic CPA2 antiporter NhaA (EcNhaA) from Escherichia coli (6) applies to the electroneutral CPA1 antiporter MjNhaP1 (7)
We found that the competition-based transport mechanism that applies to EcNhaA and MjNhaP1 describes the activity of PaNhaP
Summary
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. The study on MjNhaP1 revealed that this exchanger, overall electroneutral, has at least two electrogenic transport steps, which we assigned to the translocation of the Naϩ and Hϩ substrate ions across the membrane (7). This enabled us to monitor the activity of the transporter by solid-supported membrane (SSM)-based electrophysiology, an experimental technique that is appropriate for the characterization of prokaryotic membrane transporters (9). As in MjNhaP1, we uncovered electrogenic steps in the transport cycle of PaNhaP that effect the translocation of positive charge, both in the wild-type protein and in a mutant where Glu-73 was replaced by alanine These electrogenic steps are attributable to the binding and possibly the occlusion of the Naϩ substrate ion. Our results establish a model of substrate translocation and define the role of Glu-73 in PaNhaP
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