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 and possess 10-12 putative or established transmembrane α-helical spanners. 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). 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. Escherichia 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.
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.
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.
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.
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.
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)). 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).	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)**