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The Primary Sodium Binding Site of Human Concentrative Nucleoside Transporter 3, hCNT3 Open Access


Other title
human concentrative nucleoside transporter
cation activation kinetics
Type of item
Degree grantor
University of Alberta
Author or creator
Gawad Gad,Sandra Sabry
Supervisor and department
Young, James (Physiology)
Examining committee member and department
Cass, Carol (Oncology)
Karpinski, Edward (Physiology)
Hammond, James (Pharmacology)
Department of Physiology
Date accepted
Graduation date
Master of Science
Degree level
Nucleosides are essential for RNA and DNA synthesis. They also play a central role in other cellular metabolic pathways, and modulate a diverse array of physiological processes, including renal and cardiovascular function and neurotransmission. Due to their hydrophilic nature, specialized integral membrane proteins known as nucleoside transporters (NTs) are required for transport across cell membranes. In humans, the cation-coupled concentrative nucleoside transporter (CNT) family is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, the most functionally versatile hCNT, is a cation-nucleoside symporter that transports both purine and pyrimidine nucleosides, as well as anticancer and antiviral nucleoside drugs. Produced as a recombinant protein in the Xenopus oocyte heterologous expression system, hCNT3 has been shown to have a Na+:uridine coupling ratio of 2:1, in contrast to hCNT1/2 which have Na+:uridine coupling ratios of 1:1. One of the two Na+-binding sites of hCNT3 also accepts H+. Recently, the crystal structure of a bacterial hCNT ortholog (vcCNT from Vibrio cholerae) has been reported. Based upon the crystal structure of vcCNT and previous mutagenesis studies of hCNTs, four amino acid residues (N336, V339, T370, and I371) were postulated to coordinate Na+ (and hydronium ion) binding within the primary cation-binding site of hCNT3. To test this hypothesis, electrophysiological studies were performed on oocytes producing wild-type hCNT3 or engineered forms of the transporter in which each of the four residues were individually mutated to cysteine. The results show marked changes in Na+- and H+-coupling consistent with these residues forming the primary cation-binding site of hCNT3. Mutation of the corresponding residues in hCNT1 and characterization of wild-type and mutant forms of vcCNT in oocytes provide supporting evidence for this conclusion.
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. 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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