title: Functional analysis of acidic amino acids in the cytosolic tail of the Na+/H+ exchanger.
abstract: The mammalian Na(+)/H(+) exchanger is a membrane protein with a C-terminal regulatory cytosolic domain and an N-terminal membrane domain. Na(+)/H(+) exchanger isoform 1 (NHE1) possesses a conserved amino acid sequence of seven consecutive acidic residues in the distal region of the cytosolic tail. We examined the structural and functional role of this acidic sequence. In human NHE1, varying mutations of the sequence (753)EEDEDDD(759) resulted in defective NHE1 activity. Mutation of the core acid sequence, (755)DED(757), or of the entire sequence caused a decrease in the activity of NHE1 in response to acute acid load. This was not due to changes in Na(+) affinity but rather due to decreased maximum velocity of the protein and delayed activation. Mutation of the target sequence did not affect the ability of the cytoplasmic domain to bind carbonic anhydrase II or tescalcin but did affect calmodulin binding. Mutation of the acidic domain also caused altered sensitivity to trypsin and changes in size of the protein in gel-filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results demonstrate that the acidic sequence is critical in maintaining proper conformation of the cytosolic domain, calmodulin binding, and in maintenance of Na(+)/H(+) exchanger activity.
authors.affiliation: Department of Biochemistry, 347 Medical Science Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Xiuju
authors.initials: X
authors.lastname: Li
authors.forename: Jie
authors.initials: J
authors.lastname: Ding
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Liu
authors.forename: Brenda J
authors.initials: BJ
authors.lastname: Brix
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
chemical_list: D024342:Amino Acids, Acidic; D017923:Sodium-Hydrogen Exchangers