期刊
PROTEIN SCIENCE
卷 31, 期 12, 页码 -出版社
WILEY
DOI: 10.1002/pro.4460
关键词
human NHA2; Na+; H+ antiporter; N-terminal auto-inhibition; phloretin; yeast
资金
- Grantova Agentura.Ceske Republiky [21-08985S]
- IPHYS Mobility II [CZ.02.2.69/0.0/0.0/18_053/0016977]
- USA-Israel Binational Science Foundation
- Abraham E. Kazan Chair in Structural Biology, Tel Aviv University [2017293]
This study investigated the functional characteristics of mutated or truncated versions of HsNHA2 using a salt-sensitive Saccharomyces cerevisiae strain. The highly conserved proline 246 in the core of the protein was found to be crucial for ion selectivity. Additionally, the hydrophilic N-terminal part of the protein was shown to allosterically inhibit cation transport of HsNHA2.
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.
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