title: Structural changes in the C-terminal regulatory region of the Na⁺/H⁺ exchanger mediate phosphorylation induced regulation.
abstract: The Na(+)/H(+) exchanger isoform 1 (NHE1) is a plasma membrane pH regulatory protein that removes one intracellular H(+) in exchange for an extracellular Na(+). NHE1 is regulated by phosphorylation of the cytoplasmic regulatory region and amino acids Ser(770) and Ser(771) of the regulatory domain are necessary for activation by sustained intracellular acidosis. The phosphomimetic mutations (S770D/S771D) resulted in an activated form of the protein. Immunoprecipitation of full length NHE1 protein showed that the phosphomimetic mutant had increased sensitivity to digestion with trypsin indicating a conformational change. Tryptic digestion of purified C-terminal regulatory region showed that the S770D/S771D mutation altered sensitivity to trypsin digestion. Wild type and phosphomimetic purified C-terminal region (577-815) of human NHE1 were compared and tryptophan fluorescence indicated that there were pH-dependent differences in the conformation of the proteins. Native polyacrylamide gel electrophoresis demonstrated that the phosphomimetic mutant had a more compact structure. Bottom-up hydrogen/deuterium exchange mass spectrometry demonstrated that a peptide fragment containing the phosphomimetic mutations became strongly stabilized relative to the wild type protein. Overall, the results suggested that phosphorylation of S770/S771 changes the conformation of the C-terminal regulatory region in a pH-dependent manner, resulting in a more compact region that affects NHE1 activity. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.
authors.forename: Xiuju
authors.initials: X
authors.lastname: Li
authors.forename: Morgan F
authors.initials: MF
authors.lastname: Khan
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D019943:Amino Acid Substitution; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D041961:Deuterium Exchange Measurement; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
chemical_list: D027682:Cation Transport Proteins; D010446:Peptide Fragments; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D014357:Trypsin
keywords: Conformational change; Ion transport; Membrane protein; Na(+)/H(+) exchanger; Phosphorylation; pH regulation