Structure-function studies of novel K+ -dependent Na+/Ca2+ exchangers

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dc.contributor.advisorLytton, Jonathan
dc.contributor.authorCai, Xinjiang
dc.date.accessioned2005-08-16T16:52:08Z
dc.date.available2005-08-16T16:52:08Z
dc.date.issued2004
dc.descriptionBibliography: p. 203-244en
dc.description.abstractPlasma membrane K -dependent Na /Ca exchangers (NCKX) are believed to play a crucial role in mediating Ca2+ efflux in a wide range of tissues. This activity is encoded by a multigene family of at least five members that share sequence similarity in two internally homologous domains known as the a-repeats. The a-repeats are believed to form the ion binding sites for translocation. I have explored the structure-function relationship of the rat brain NCKX2 exchanger. Two important Cys residues were identified to be critical for stabilizing protein structure. Using an introduced epitope I demonstrated that the protein C-terminus was extracellularly exposed in intact cells. A new topological model for NCKX2 has been presented, which is different from the initial model for NCKX exchangers. NCKX2 could be labeled with biotin maleimide only following application of (3-mercaptoethanol. NCKX2 activity was also enhanced by (3-mercaptoethanol. A critical residue Cys-395 in the large intracellular loop was found to underlie the redox-dependent labeling and functional stimulation. Although NCKX2 associates as an oligomer, this interaction does not require Cys-395. I have isolated a novel cDNA clone encoding NCKX6 from mouse thymus. NCKX6 is a structurally more divergent, and distantly related, Na+/Ca2+ exchanger member compared with other known exchangers. Identification of many NCKX6 paralogs from lower organisms suggested NCKX6 might have evolved independently from the other NCKX members in the Na /Ca exchanger superfamily. A splice variant with a truncated C-terminal hydrophobic exchanger domain, but not the full-length clone, could reach the plasma membrane when expressed in HEK-293 cells. This NCKX6 isoform exhibited K+-dependent Na+/Ca2+ exchange activity. Immunofluorescence staining using a polyclonal NCKX6-specific antibody demonstrated strong fluorescence labeling at the cell surface in native cells. The full-length NCKX6 and its splice variants are expressed in every tissue examined and their relative expression levels were compared. I have performed a comprehensive bioinformatic analysis of the cation/Ca2+ exchanger superfamily, integrating genomic, functional, structural and evolutionary information. Our data defined five major protein families, each with a distinct evolutionary relationship and unique signature motifs. These findings may provide guides for future studies concerning the structure-to-function relationship and evolutionary origins of the cation/Ca2+ exchangers.en
dc.format.extentxv, 292 leaves : ill. ; 30 cm.en
dc.identifier.citationCai, X. (2004). Structure-function studies of novel K+ -dependent Na+/Ca2+ exchangers (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/21471en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/21471
dc.identifier.isbn0494039019en
dc.identifier.lccAC1 .T484 2004 C35en
dc.identifier.urihttp://hdl.handle.net/1880/41382
dc.language.isoeng
dc.publisher.institutionUniversity of Calgaryen
dc.publisher.placeCalgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.titleStructure-function studies of novel K+ -dependent Na+/Ca2+ exchangers
dc.typedoctoral thesis
thesis.degree.disciplineCardiovascular & Respiratory Sciences
thesis.degree.grantorUniversity of Calgary
thesis.degree.nameDoctor of Philosophy (PhD)
ucalgary.item.requestcopytrue
ucalgary.thesis.accessionTheses Collection 58.002:Box 1493 520492010
ucalgary.thesis.notesUARCen
ucalgary.thesis.uarcreleaseyen
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