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). The crossing of two unwound transmembrane regions that is the hallmark of the NhaA structural fold is critical for antiporter activity (Rimon et al. 2024). 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:
Appel, M., D. Hizlan, K.R. Vinothkumar, C. Ziegler, and W. Kühlbrandt. (2009). Conformations of NhaA, the Na/H exchanger from Escherichia coli, in the pH-activated and ion-translocating states. J. Mol. Biol. 386: 351-365.
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.
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.
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.
Diab, M., A. Rimon, T. Tzubery, and E. Padan. (2011). Helix VIII of NhaA Na⁺/H⁺ antiporter participates in the periplasmic cation passage and pH regulation of the antiporter. J. Mol. Biol. 413: 604-614.
Dwivedi, M. (2020). Site-directed mutations reflecting functional and structural properties of Ec-NhaA. Biochimie 180: 79-89. [Epub: Ahead of Print]
Gerchman, Y., Y. Olami, A. Romon, D. Taglicht, S. Schuldiner, and E. Padan (1993). Histidine-226 is part of the pH sensor of NhaA, a Na⁺:H⁺ antiporter in Escherichia coli. Proc. Natl. Acad. Sci. USA 90: 1212-1216.
Herz, K., A. Rimon, E. Olkhova, L. Kozachkov, and E. Padan. (2010). Transmembrane segment II of NhaA Na⁺/H⁺ antiporter lines the cation passage, and Asp65 is critical for pH activation of the antiporter. J. Biol. Chem. 285: 2211-2220.
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.
Karpel, R., Y. Olami, D. Taglicht, S. Schuldiner, and E. Padan (1988). Sequencing of the gene ant which affects the Na⁺:H⁺ antiporter activity in Escherichia coli. J. Biol. Chem. 263: 10408-10414.
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.
Kozachkov, L., K. Herz, and E. Padan. (2007). Functional and structural interactions of the transmembrane domain X of NhaA, Na⁺/H⁺ antiporter of Escherichia coli, at physiological pH. Biochemistry 46: 2419-2430.
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.
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.
Padan, E. (2008). The enlightening encounter between structure and function in the NhaA Na⁺-H⁺ antiporter. Trends. Biochem. Sci. 33: 435-443.
Padan, E., M. Venturi, Y. Gerchman, and N. Dover. (2001). Na⁺/H⁺ antiporters. Biochim. Biophys. Acta 1505: 144-157.
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.
Patiño-Ruiz, M., C. Ganea, and O. Călinescu. (2022). Prokaryotic Na/H Exchangers-Transport Mechanism and Essential Residues. Int J Mol Sci 23:.
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.
Rimon, A., H. Amartely, and E. Padan. (2024). The crossing of two unwound transmembrane regions that is the hallmark of the NhaA structural fold is critical for antiporter activity. Sci Rep 14: 5915.
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.
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.
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.
Taglicht, D., E. Padan, and S. Schuldiner. (1991). Overproduction and purification of a functional Na⁺:H⁺ antiporter coded by NhaA (ant) from Escherichia coli. J. Biol. Chem. 266: 11289-11294.
White, N., H. Sadeeshkumar, A. Sun, N. Sudarsan, and R.R. Breaker. (2022). Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes. Sci Rep 12: 19145.
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.
Williams, K.A. (2000). Three-dimensional structure of the ion-coupled transport protein NhaA. Nature 403: 112-115.
Examples:
TC#	Name	Organismal Type	Example
2.A.33.1.1	NhaA Na⁺:2H⁺ antiporter (structure determined and mechanism proposed (Williams, 2000; Hunte et al., 2005; Olkhova et al., 2006; Screpanti et al., 2006; Arkin et al., 2007)). The K300R mutant is also electrogenic (Călinescu et al. 2017). TMS II lines the cation passage, and Asp65 is critical for pH activation of the antiporter (Herz et al., 2010). NhaA is subject to pH-activation of the ion-translocating conformation (Appel et al., 2009; Diab et al., 2011). A periplasm-facing state of the NhaA antiporter suggests the molecular underpinnings of the pH-induced conformational changes (Schushan et al., 2012). A central unwound part of TMS IV appears to line the cation passage channel (Rimon et al. 2012). TMSs VI and VII are absent from many homologues, are not required for transport or its regulation and function in assembly and stability (Padan et al. 2015). pH-induced conformational changes have been documented (Kozachkov et al. 2007). Two acidic residues in the binding site that carries the protons in electrogenic CPAs, and a polar residue in the unwound transmembrane helix 4 that determines ion selectivity have been identified. A rationally designed triple mutant successfully converted the electrogenic EcNhaA, to be electroneutral (Masrati et al. 2018). Residues involved in NhaA function have been reviewed (Dwivedi 2020). The type of residues involved in substrate binding and even a simple mechanism sufficient to explain the pH regulation in the CPA and IT superfamilieshave been reviewed (Patiño-Ruiz et al. 2022). Several aspects of prokaryotic Na⁺/H⁺ exchanger structures and function are presented, discussing the similarities and differences between different transporters, with a focus on the CPA and IT exchangers (Patiño-Ruiz et al. 2022).	Gram-negative bacteria	NhaA of E. coli

2.A.33.1.2	*NhaA Na⁺,K⁺:H⁺ antiporter (Radchenko et al., 2006)*	Gram-negative bacteria	*NhaA of Vibrio parahaemolyticus* (BAC59491)**

2.A.33.1.3	Na⁺/H⁺ antiporter NhaA (Sodium/proton antiporter NhaA)	Bacteria	NhaA of Geobacter metallireducens

2.A.33.1.4	Na⁺/H⁺ antiporter NhaA 1 (Sodium/proton antiporter NhaA 1)	Bacteria	NhaA1 of Pelobacter propionicus

2.A.33.1.5	Na⁺/H⁺ antiporter (Sodium/proton antiporter), NhaA	Bacteria	NhaA of Bifidobacterium longum

2.A.33.1.6	Na⁺/H⁺ antiporter (Sodium/proton antiporter), NhaA	Bacteria	NhaA1 of Streptomyces coelicolor / M145)

2.A.33.1.7	Na⁺or Ca²⁺/H⁺ antiporter, NhaA of 492 aas and 12 TMSs, active at alkaline pH. Confers tolerance to high NaCl concentrations. Has a Km_Na⁺ of 0.2 - 1 mM, and a Km_Ca²⁺ of 0.1 - 0.3 mM (Dawut et al. 2018).		NhaA of the marine picoalga, Ostreococcus tauri.