Group in Protein Structure and Function, and ¶Protein Engineering Network of Centers of Excellence,

The Na/H exchanger isoform 1 is a ubiquitously ex-pressed integral membrane protein that regulates intracel-lular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) seg-ment IV. Each residue was mutated to cysteine in cysteine-less NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23mutations causing greatly reduced expression and/or activity. The Phe1613 Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive com-pounds [2-(trimethylammonium)ethyl]methanethiosulfo-nate and [2-sulfonatoethyl]methanethiosulfonate, suggest-ing it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD3OH:CDCl3:H2O mixture and in Me2SO. In CD3OH: CDCl3:H2O, TM IV was structured but not as a canonica