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 protein that regulates intracellular pH in mammalian cells. Site-specific mutagenesis was used to examine the functional role of conserved, polar amino-acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino-acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of 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 mutated 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 support the hypothesis that side-chain oxygen atoms in a few, critically placed amino 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 involved in Na+ coordination by the protein.
Murtazina
R
R
Department of Biochemistry, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada.
Booth
B J
BJ
Bullis
B L
BL
Singh
D N
DN
Fliegel
L
L
D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002412:Cations / Q000378:metabolism; D006224:Cricetinae; D017136:Ion Transport; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D002412:Cations; D008565:Membrane Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D014357:Trypsin