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Permanent link (DOI): https://doi.org/10.7939/R3CD25

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Functional studies on the human sodium proton exchanger isoform 1 Open Access

Descriptions

Other title
Subject/Keyword
NHE1
Pore
MTSET
Transmembrane
Cysteine
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Tzeng, Jennifer
Supervisor and department
Fliegel, Larry (Biochemistry)
Examining committee member and department
Sykes, Brian (Biochemistry)
Casey, Joe (Physiology)
Department
Department of Biochemistry
Specialization

Date accepted
2011-04-14T21:59:18Z
Graduation date
2011-06
Degree
Master of Science
Degree level
Master's
Abstract
The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous membrane protein that exchanges one intracellular H+ for an extracellular Na+, thereby regulating cell pH and volume. NHE1 catalytic activity is mediated by a transmembrane (TM) domain with 12 transmembrane segments. We performed cysteine scanning mutagenesis on TMVI (Asn227–Ile249) of NHE1. Each residue of TMVI was mutated into a cysteine in the background of a cysteineless NHE1 protein. MTSET and MTSES are sulfhydryl reactive membrane impermeable compounds able to react with accessible cysteines. Asp238Cys, Pro239Cys, and Glu247Cys expressed inactive NHE1. Asn227Cys, Ile233Cys, and Leu243Cys were strongly inhibited by MTSET, suggesting their pore lining properties. More mutations were introduced to characterize critical residues in TMVI. The Glu248Gln and Leu243Ala mutants were more susceptible to limited proteolytic attack by trypsin suggesting an altered conformation. The results suggest that Glu248 and Leu243 are important in protein structure, stability, and folding.
Language
English
DOI
doi:10.7939/R3CD25
Rights
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.
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File format: pdf (Portable Document Format)
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File author: Larry Fliegel
Page count: 108
File language: en-CA
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