Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane pr otein that regulates intracellular pH in mammalian cells. Site-specific mut agenesis was used to examine the functional role of conserved, polar amino- acid residues occurring in segments of the protein associated with the memb rane. Seventeen mutant proteins were assessed by characterization of intrac ellular pH changes in stably transfected cells that lacked an endogenous Na +/H+ exchanger. All of the mutant proteins were targeted correctly to the p lasma membrane and were expressed at similar levels. Amino-acid residues Gl u262 and Asp267 were critical to Na+/H+ exchanger activity while mutation o f Glu391 resulted in only a partial reduction in activity. The Glu262-->Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutate d Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H + exchanger activity. The Glu262-->Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results suppo rt the hypothesis that sidechain oxygen atoms in a few, critically placed a mino acids are important in Na+/H+ exchanger activity and the acidic amino- acid residues at positions 262, 267 and 391 are good candidates for being i nvolved in Na+ coordination by the protein.