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2.A.33 The NhaA Na+:H+Antiporter (NhaA) Family

NhaA homologues have been sequenced from numerous bacteria and archaea. Many prokaryotes possess multiple paralogues. These proteins are of 300-700 amino acyl residues in length. The E. coli protein probably functions in the regulation of the internal pH when the external pH is alkaline, and the protein effectively functions as a pH sensor (Gerchman et al., 1993). It also uses the H+ gradient to expel Na+ from the cell. Its activity is highly pH dependent. Only the E. coli protein is functionally and structurally well characterized (Padan et al., 2001; Hunte et al., 2005). Its structure reveals a homeodimer, each subunit consisting of a bundle of 12 tilted transmembrane α-helices (Williams et al., 1999; Williams, 2000; Hunte et al., 2006; Olkhova et al., 2006; Screpanti et al., 2006).

Molecular dynamics simulations of NhaA enabled proposal of an atomically detailed model of antiporter function Arkin et al., 2007). Three conserved aspartates are key to this proposed mechanism: Asp164 (D164) is the Na+-binding site, D163 controls the alternating accessibility of this binding site to the cytoplasm or periplasm, and D133 is crucial for pH regulation. Consistent with experimental stoichiometry, two protons are required to transport a single Na+ ion: D163 protonates to reveal the Na+-binding site to the periplasm, and subsequent protonation of D164 releases Na+ (Arkin et al., 2007; Padan, 2008). A Trp at position 136 specifically monitors a pH-induced conformational change that activates NhaA, wheras a Trp at position 339 senses a ligand-induced conformational change that does not occur until NhaA is activated at alkaline pH (Kozachkov and Padan, 2011).The primary function of Li+ riboswitches and associated NhaA transporters is to prevent Li+ toxicity, particularly when bacteria are living at high pH (White et al. 2022).

Na+-H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic membrane and many intracellular membranes. They are essential for Na+, pH, and volume homeostasis, which are processes crucial for cell viability. Accordingly, antiporters are important drug targets in humans and underlie salt resistance in plants. Many Na+-H+ antiporters are tightly regulated by pH. E. coli NhaA, a prototype pH-regulated antiporter, exchanges 2H+ for 1Na+ (or Li+). The NhaA crystal structure has provided insight into the pH-regulated mechanism of antiporter action and revealed transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found with variations in other ion-transport proteins. This novel structural fold creates a delicately balance electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation.

The generalized transport reaction catalyzed by NhaA is:

Na+ (in) + 2H+ (out) ⇌ Na+ (out) + 2H+ (in)

References associated with 2.A.33 family:

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Arkin, I.T., H. Xu, M.Ø. Jensen, E. Arbely, E.R. Bennett, K.J. Bowers, E. Chow, R.O. Dror, M.P. Eastwood, R. Flitman-Tene, B.A. Gregersen, J.L. Klepeis, I. Kolossváry, Y. Shan, and D.E. Shaw. (2007). Mechanism of Na+/H+ antiporting. Science. 317: 799-803. 17690293
Călinescu, O., M. Dwivedi, M. Patiño-Ruiz, E. Padan, and K. Fendler. (2017). Lysine 300 is essential for stability but not for electrogenic transport of the NhaA Na/H antiporter. J. Biol. Chem. 292: 7932-7941. 28330875
Dawut, K., S. Sirisattha, T. Hibino, H. Kageyama, and R. Waditee-Sirisattha. (2018). Functional characterization of the NhaA Na/H antiporter from the green picoalga Ostreococcus tauri. Arch Biochem Biophys 649: 37-46. 29730321
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Hunte, C., E. Screpanti, M. Venturi, A. Rimon, E. Padan, and H. Michel. (2005). Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH. Nature. 435: 1197-1202. 15988517
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Kozachkov, L. and E. Padan. (2011). Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na+/H+ antiporter NhaA. Proc. Natl. Acad. Sci. USA 108: 15769-15774. 21873214
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Masrati, G., M. Dwivedi, A. Rimon, Y. Gluck-Margolin, A. Kessel, H. Ashkenazy, I. Mayrose, E. Padan, and N. Ben-Tal. (2018). Broad phylogenetic analysis of cation/proton antiporters reveals transport determinants. Nat Commun 9: 4205. 30310075
Olkhova, E., C. Hunte, E. Screpanti, E. Padan, and H. Michel. (2006). Multiconformation continuum electrostatics analysis of the NhaA Na+/H+ antiporter of Escherichia coli with functional implications. Proc. Natl. Acad. Sci. U.S.A. 103: 2629-2634. 16477015
Padan, E. (2008). The enlightening encounter between structure and function in the NhaA Na+-H+ antiporter. Trends. Biochem. Sci. 33: 435-443. 18707888
Padan, E., M. Venturi, Y. Gerchman, and N. Dover. (2001). Na+/H+ antiporters. Biochim. Biophys. Acta 1505: 144-157. 11248196
Padan, E., T. Danieli, Y. Keren, D. Alkoby, G. Masrati, T. Haliloglu, N. Ben-Tal, and A. Rimon. (2015). NhaA antiporter functions using 10 helices, and an additional 2 contribute to assembly/stability. Proc. Natl. Acad. Sci. USA 112: E5575-5582. 26417087
Radchenko, M.V., R. Waditee, S. Oshimi, M. Fukuhara, T. Takabe, and T. Nakamura. (2006). Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli. Mol. Microbiol. 59: 651-663. 16390457
Rimon, A., L. Kozachkov-Magrisso, and E. Padan. (2012). The unwound portion dividing helix IV of NhaA undergoes a conformational change at physiological pH and lines the cation passage. Biochemistry 51: 9560-9569. 23131124
Schushan, M., A. Rimon, T. Haliloglu, L.R. Forrest, E. Padan, and N. Ben-Tal. (2012). A Model-Structure of a Periplasm-facing State of the NhaA Antiporter Suggests the Molecular Underpinnings of pH-induced Conformational Changes. J. Biol. Chem. 287: 18249-18261. 22431724
Screpanti, E., E. Padan, A. Rimon, H. Michel, and C. Hunte. (2006). Crucial steps in the structure determination of the Na+/H+ antiporter NhaA in its native conformation. J. Mol. Biol. 362: 192-202. 16919297
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Williams, A., U. Geldmacher-Kaufer, E. Padan, S. Schuldiner, and W. Kühlbrandt. (1999). Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0Å resolution. EMBO J. 18: 3558-3563. 10393172
Williams, K.A. (2000). Three-dimensional structure of the ion-coupled transport protein NhaA. Nature 403: 112-115. 10638764