Structural and functional characterization of transmembrane segment IV of the NHE1 isoform of the Na+/H+ exchanger

Emily R. Slepkov, Jan K. Rainey, Xiuju Li, Yongsheng Liu, Florence J. Cheng, Darrin A. Lindhout, Brian D. Sykes, Larry Fliegel

Research output: Contribution to journalArticlepeer-review

84 Citations (Scopus)

Abstract

The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteineless NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe 161 → Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylanunonium)ethyl] methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD3OH:CBCl3:H2O mixture and in Me2SO. In CD3OH: CDCl3:H 2O, TM IV was structured but not as a canonical α-helix. Residues Asp159-Leu162 were a series of β-turns; residues Leu165-Pro168 showed an extended structure, and residues Ile169-Phe176 were helical in character. These three structured regions rotated quite freely with respect to the others. In Me2SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe161 is a pore-lining residue.

Original languageEnglish
Pages (from-to)17863-17872
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number18
DOIs
Publication statusPublished - May 6 2005
Externally publishedYes

ASJC Scopus Subject Areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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