ERA

Download the full-sized PDF of Substituted Cysteine Accessibility Method Analysis of the C-terminal Half of Human Concentrative Nucleoside Transporter 3 (hCNT3)Download the full-sized PDF

Analytics

Share

Permanent link (DOI): https://doi.org/10.7939/R3SX22

Download

Export to: EndNote  |  Zotero  |  Mendeley

Communities

This file is in the following communities:

Graduate Studies and Research, Faculty of

Collections

This file is in the following collections:

Theses and Dissertations

Substituted Cysteine Accessibility Method Analysis of the C-terminal Half of Human Concentrative Nucleoside Transporter 3 (hCNT3) Open Access

Descriptions

Other title
Subject/Keyword
hCNT3 SCAM Analysis
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Mulinta, Ras
Supervisor and department
Young, James D. (Physiology)
Examining committee member and department
Cass, Carol E. (Oncology)
Casey, Joseph (Physiology)
Landfear, Scott (Oregon Health & Science University, Molecular Microbiology & Immunology)
Cheeseman, Christopher (Physiology)
Duszyk, Marek (Physiology)
Department
Department of Physiology
Specialization

Date accepted
2010-01-15T19:37:13Z
Graduation date
2010-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Concentrative nucleoside transporter (CNT) proteins mediate active nucleoside transport using the electrochemical gradient of the coupling cation. The molecular mechanisms underlying interactions with both nucleosides and cations were investigated by heterelogous expression of recombinant CNT family members in Xenopus oocytes. Substituted cysteine accessibility method (SCAM) analysis in combination with radioisotope flux assays and electrophysiological studies revealed novel topological features within the C-terminal half of human (h)CNTs and identified residues of functional importance. The hCNT (SLC28) protein family is represented by three members. hCNT1 and hCNT2 are pyrimidine nucleoside- and purine nucleoside-selective, respectively, while hCNT3 transports both pyrimidine and purine nucleosides. hCNT1 and hCNT2 function exclusively as Na+-coupled nucleoside transporters and share a 1:1 Na+:nucleoside stoichiometry. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of Na+, Li+ or H+ to drive nucleoside transport and exhibits 2:1 Na+:nucleoside and 1:1 H+:nucleoside stoichiometries. Non-mammalian H+-coupled CNT family members that have been functionally characterized include NupC from Escherichia coli. Both Na+ and H+ activate CNTs through mechanisms to increase nucleoside apparent binding affinity. Multiple alignments of CNT family members reveal strong sequence similarities within the C-terminal halves of the proteins, and hCNT1/3 and other chimeric studies have demonstrated that this region determines both nucleoside and cation interactions with the transporter. In hCNT3, access of pchloromercuribenzene sulfonate (PCMBS) to introduced cysteine residues within putative transmembrane segments (TMs) 7, 8, 9 and 11A revealed novel discontinuous regions within α-helical structures, whereas putative TMs 10, 11, 12 and 13 exhibited conventional α-helical characteristics. Putative TM 11A, which contains the highly conserved CNT family motif (G/A)XKX3NEFVA(Y/M/F), was shown to be membrane associated and, most likely, membrane spanning, TMs 7-11 having a reversed orientation in the membrane compared to previous models of CNT topology. Furthermore, putative TMs 7, 8, 9, 11A and 12 were shown to contribute functional and structural elements to a common nucleoside/cation translocation pore. These studies, which were extended to TMs 7 and 8 of hCNT1 and to corresponding TMs of E. coli NupC, provide important structural and functional insights into the nature of CNT nucleoside/cation cotransport.
Language
English
DOI
doi:10.7939/R3SX22
Rights
License granted by Ras Mulinta (mras@ualberta.ca) on 2010-01-15 (GMT): 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 the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein 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

File Details

Date Uploaded
Date Modified
2014-05-01T00:27:17.529+00:00
Audit Status
Audits have not yet been run on this file.
Characterization
File format: pdf (Portable Document Format)
Mime type: application/pdf
File size: 4891521
Last modified: 2015:10:12 14:09:57-06:00
Filename: Mulinta_Ras_Winter 2010.pdf
Original checksum: a513e558a4f142fa46b0ef0f261dbc7b
Well formed: true
Valid: true
Status message: Too many fonts to report; some fonts omitted. Total fonts = 1169
File title: Ras Front pages of Thesis after defense 13.1.10.pdf
File title: University of Alberta
File author: Ras Mulinta
Page count: 334
Activity of users you follow
User Activity Date