Structural and Functional Characterization of Transmembrane

TheNa/H exchanger isoform1 (NHE1) is an integralmem-brane protein that regulates intracellular pH by removing one intracellular H in exchange for one extracellular Na. It has a large N-terminal membrane domain of 12 transmembrane seg-ments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339–363). Each residue wasmutated to cysteine in a functional cysteine-less NHE1 protein. Of 25 amino acids mutated, 5 were inac-tive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 function. Of the active mutants, Glu346 and Ser351 were inhib-ited>70 % by positively charged [2- (trimethylammonium)