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

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.