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


Other title
Type of item
Degree grantor
University of Alberta
Author or creator
Kemp, Grant A.
Supervisor and department
Young, Howard (Biochemistry)
Fliegel, Larry (Biochemistry)
Examining committee member and department
Walsh, Michael (Biochemistry and Molecular Biology, University of Calgary)
Young, James (Physiology)
Lemieux, M. Joanne (Biochemistry)
Department of Biochemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
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.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Karine Moncoq, Grant Kemp, Xiuju Li, Larry Fliegel and Howard S. Young. Dimeric Structure of Human Na+ /H+ Exchanger Isoform 1 Overproduced in Saccharomyces cerevisiae. J. Biol. Chem. 2008; 283:4145–4154.Asad Ullah, Grant Kemp, Brian Lee, Claudia Alves, Howard S. Young, Brian D. Sykes and Larry Fliegel. Structural and Functional Analysis of Transmembrane Segment IV of the Salt Tolerance Protein Sod2. J. Biol. Chem. 2013; 288(34):24609–24624.Grant Kemp, Howard S. Young, and Larry Fliegel. Structure and function of the human Na+ /H+ exchanger isoform 1. Channels (Austin). 2008. 2: 329–336.

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