2.A.34 The NhaB Na⁺:H⁺ Antiporter (NhaB) Family NhaB homologues are of 513 to 528 amino acyl residues in length and possess about 12 transmembrane α-helical spanners (TMSs). They exhibit a region with limited sequence similarity to a 46 Kd membrane protein of unknown function from Mycobacterium leprae (spP46838) which is homologous to a member of arsenate resistance pumps of bacteria, archaea and eukaryotes (TC #3.A.4). Members of the Cation/Proton Antiporter (CPA) superfamily, the Ion Transport (IT) superfamily, and the Na⁺-translocating Mrp transporter superfamily can catalyze Na+/H+ antiiport (Patiño-Ruiz et al. 2022). Transport mechanisms for Na⁺/H⁺ exchangers that explain their highly pH-regulated activity profiles have been considered (Patiño-Ruiz et al. 2022). The E. coli NhaB is 58% identical to the orthologous Vibrio alginolyticus Na⁺/H⁺ antiporter (Pinner et al., 1992). Although the latter protein is predicted to exhibit 10 TMSs, construction of NhaB-phoA fusions led to evidence for a 9 TMS model with the N-terminus in the cytoplasm and the C-terminus in the periplasm (Enomoto et al., 1998). A centrally located aspartyl residue in the 3rd TMS of the Vibrio alginolyticus homologue, conserved in all members of the family, has been shown to be essential for activity (Nakamura et al., 2001). The Klebsiella pneumoniae ortholog has also been studied in some detail, revealing stoichiomentric ratios in the wild type and mutant proteins (Patiño-Ruiz et al. 2017; Patiño-Ruiz et al. 2019). The generalized transport reaction catalyzed by NhaB of E. coli is: 2 Na⁺ (in) + 3 H⁺ (out) ⇌ 2 Na⁺ (out) + 3 H⁺ (in)
This family belongs to the IT Superfamily.
References:
Enomoto, H., T. Unemoto, M. Nishibuchi, E. Padan, and T. Nakamura. (1998). Topological study of the Vibrio alginolyticus Na⁺/H⁺ antiporter using gene fusions in Escherichia coli cells. Biochim. Biophys. Acta 1370: 77-86.
Nakamura, T., Y. Fujisaki, H. Enomoto, Y. Nakayama, T. Takabe, N. Yamaguchi, and N. Uozumi. (2001). Residue aspartate-147 from the third transmembrane region of Na⁺/H⁺ antiporter NhaB of Vibrio alginolyticus plays a role in its activity. J. Bacteriol. 183: 5762-5767.
Patiño-Ruiz, M., C. Ganea, and O. Călinescu. (2022). Prokaryotic Na/H Exchangers-Transport Mechanism and Essential Residues. Int J Mol Sci 23:.
Patiño-Ruiz, M., C. Ganea, K. Fendler, and O. Călinescu. (2017). Competition is the basis of the transport mechanism of the NhaB Na⁺/H⁺ exchanger from Klebsiella pneumoniae. PLoS One 12: e0182293.
Patiño-Ruiz, M., K. Fendler, and O. Călinescu. (2019). Mutation of two key aspartate residues alters stoichiometry of the NhaB Na/H exchanger from Klebsiella pneumoniae. Sci Rep 9: 15390.
Pinner, E., E. Padan, and S. Schuldiner. (1992). Cloning, sequencing and expression of the NhaB gene, encoding a Na⁺:H⁺ antiporter in Escherichia coli. J. Biol. Chem. 267: 11064-11068.
Pinner, E., E. Padan, and S. Schuldiner. (1994). Kinetic properties of NhaB, a Na⁺/H⁺ antiporter from Escherichia coli. J. Biol. Chem. 269: 26274-26279.
Pinner, E., Y. Kotler, E. Padan, and S. Schuldiner. (1993). Physiological role of nhaB, a specific Na⁺/H⁺ antiporter in Escherichia coli. J. Biol. Chem. 268: 1729-1734.
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.
Shimamoto, T., K. Inaba, P. Thelen, T. Ishikawa, E.B. Goldberg, M. Tsuda, and T. Tsuchiya. (1994). The NhaB Na⁺/H⁺ antiporter is essential for intracellular pH regulation under alkaline conditions in Escherichia coli. J Biochem 116: 285-290.
Examples:
TC#	Name	Organismal Type	Example
2.A.34.1.1	NhaB Na⁺:H⁺ antiporter of 513 aas and 11 or 12 TMSs. NhaB is physiologically important when the level of NhaA activity is growth limiting, when nhaA is not sufficiently induced, and/or when NhaA is not activated (Pinner et al. 1993). The stoichiometry of NhaB is 3H⁺/2Na⁺, different from that of NhaA (Pinner et al. 1994). It is required for intracellular pH regulation under alkaline conditions (Shimamoto et al. 1994). Mutations D146A/D404A in the Klebsiella pneumoniae ortholog changed the stoichiometry to a H⁺:Na⁺ ratio of < 1 (Patiño-Ruiz et al. 2019).	Bacteria and archaea	*NhaB of E. coli* (P0AFA7)**

2.A.34.1.2	*NhaB Na⁺,K⁺:H⁺ antiporter (Radchenko et al., 2006)*	Bacteria and archaea	*NhaB of Vibrio parahaemolyticus* (BAC60335)**

2.A.34.1.3	Na⁺/H⁺ antiporter that extrudes sodium in exchange for external protons, NhaB of 500 aas. Can also extrude lithium in exchange for protons. Km values for Li⁺ and Na⁺ of 2.0 mM and 1.3 mM, respectively. The antiport activity was inhibited by amiloride with a Ki value of 0.04 mM (Kuroda et al. 2004).		NhaB of Pseudomonas aeruginosa