Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2

Diego Velázquez, Vojtěch Průša, Gal Masrati, Elon Yariv, Hana Sychrova, Nir Ben-Tal, Olga Zimmermannova*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


The human Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2-specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50–70 residues of the N-terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically auto-inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity.

Original languageEnglish
Article numbere4460
JournalProtein Science
Issue number12
StatePublished - Dec 2022


FundersFunder number
United States - Israel Binational Science Foundation
Grantová Agentura České Republiky2017293, CZ.02.2.69/0.0/0.0/18_053/0016977, 21‐08985S
Tel Aviv University


    • N-terminal auto-inhibition
    • Na/H antiporter
    • human NHA2
    • phloretin
    • yeast


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