title: The Na+/H+ exchanger cytoplasmic tail: structure, function, and interactions with tescalcin.
abstract: We characterized the regulatory cytoplasmic tail of the Na(+)/H(+) exchanger using a histidine-tagged protein containing the C-terminal 182 amino acids (His182). Both tescalcin and calmodulin, two Na(+)/H(+) exchanger binding proteins, bound to the His182 protein. Cascade blue was used to label the His182 protein. Calcium caused an increase in fluorescence, suggesting exposure of the label on the protein to a more hydrophilic environment. Decreasing external pH caused a transient increase in cascade blue fluorescence, followed by a decrease in fluorescence of the cascade blue labeled Na(+)/H(+) exchanger C-terminus. Tescalcin caused a decrease in fluorescence by labeled His182 protein, and calcium reversed this effect. Expression of tescalcin in vivo inhibited activity of the Na(+)/H(+) exchanger when there was an intact C-terminus of the protein. We examined the CD spectra of His182 in the presence and absence of tescalcin. The C-terminal amino acids demonstrated a very small amount of alpha-helical structure and much more beta-sheet and beta-turn. This was not greatly affected by the presence of tescalcin, but calcium caused an increase in the amount of beta-structure and a decrease in the unstructured proportion of the protein. Sedimentation equilibrium analysis demonstrated that the C-terminal 182 amino acids exist predominantly as a monomer. The results suggest that the C-terminus of the Na(+)/H(+) exchanger exists primarily as a monomeric protein that binds regulatory tescalcin and can change conformation depending on pH and calcium. Conformation changes in this region of the protein may be responsible for altering the pH sensitivity of the intact Na(+)/H(+) exchanger.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, CIHR Membrane Protein Research Group, University of Alberta, 347 Medical Science Building, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Xiuju
authors.initials: X
authors.lastname: Li
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Liu
authors.forename: Cyril M
authors.initials: CM
authors.lastname: Kay
authors.forename: Werner
authors.initials: W
authors.lastname: Müller-Esterl
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D009942:Organometallic Compounds / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D002135:Calcium-Binding Proteins; D002147:Calmodulin; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; C420458:Tesc protein, mouse; C066850:Cascade Blue; D006639:Histidine; D008274:Magnesium; D002118:Calcium