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Towards a Deeper Structural Understanding of Eukaryotic Na+/H+ Exchangers

  • Author / Creator
    Kemp, Grant A.
  • Sodium proton exchangers (NHEs) are polytopic membrane proteins that, in archaea, bacteria, yeast and plants, provide increased salt tolerance by removing excess toxic sodium, and in mammals regulate cell volume, growth, differentiation, proliferation, migration and apoptosis in relation to changes in either pH or sodium concentration. As an essential player in cellular physiology it is not surprising that NHE1 dysregulation in the body has been implicated in several diseases, which result from pathological regulation of NHE1 activity. Indeed, the scarcity of structural data has prevented the elucidation of a precise molecular mechanism of ion transport and despite recent technical advances, only a small handful of eukaryotic membrane protein structures have been uncovered. Herein are presented our advances in developing and optimizing an expression system for producing both a full-length human NHE1 protein and larger portions of the transmembrane domain thought to be responsible for ion transport. The three dimensional molecular envelope of human NHE1 was determined by single particle reconstruction electron microscopy, and progress towards determining the structure of transmembrane segments V-VII has been completed by NMR. The data presented in this thesis contribute to an improved understanding of NHE1 function at the molecular level and will help inform future therapeutic development. Additionally, I present my contribution towards characterizing transmembrane segment IV (TM IV) of sod2, a primary sodium proton exchanger in Schizosaccharomyces pombe. Functional analysis of TM IV have uncovered that Thr144–Val147 are critical to competent ion transport and the structural basis for this functional effect was analyzed by NMR, revealing a partially unwound helical conformation of TM IV in the centre of the membrane. To better hypothesize what role TM IV may play in the full length protein, we created a homology model of sod2, which indicated that sod2 TM IV is likely analogous to E. coli NhaA TM IV and human NHE1 TM VI. This study further confirmed the importance of partially unwound helices in the transport mechanism of sodium proton exchangers and provides a basis for further experimentation.

  • Subjects / Keywords
  • Graduation date
    2013-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3BQ2Z
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • Fliegel, Larry (Biochemistry)
    • Young, Howard (Biochemistry)
  • Examining committee members and their departments
    • Lemieux, M. Joanne (Biochemistry)
    • Walsh, Michael (Biochemistry and Molecular Biology, University of Calgary)
    • Young, James (Physiology)