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Towards a Structural Model of the Plasma Membrane Cl-/HCO3- Exchanger, AE1 Open Access


Other title
Towards a Structural Model of AE1
AE1 structure
homology model
Bicarbonate transport
protein expression and purification
AE1 physiology
dimer interface
Type of item
Degree grantor
University of Alberta
Author or creator
Bonar, Pamela T
Supervisor and department
Casey, Joseph (Biochemistry and Physiology)
Examining committee member and department
Glover, Mark (Biochemistry)
Alper, Seth (Harvard Medical School)
Lemieux, Joanne (Biochemistry)
Cordat, Emannuelle (Physiology)
Michalak, Marek (Biochemistry)
Department of Biochemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
AE1 is an electroneutral Cl-/HCO3- exchanger expressed in erythrocytes and the renal collecting duct. There is no high-resolution structure of the membrane domain, which alone is required for the transport activity of AE1. Here, a Saccharomyces cerevisiae expression and immuno-affinity purification system was developed for the AE1 membrane domain. The human AE1 membrane domain (residues 388-911), followed by a rhodopsin antibody epitope (AE1MD-Rho), was expressed at 0.3 mg/l of culture, and milligram quantities were purified to 93% homogeneity. AE1MD-Rho transport activity was indistinguishable from erythrocyte AE1, as assessed by radioactive [35S]SO42- efflux assays in reconstituted proteoliposomes. More recently, an electron microscopy structure of the AE1 membrane domain was proposed to have a similar protein fold to ClC chloride channels. A three-dimensional homology model of the AE1 membrane domain was created, using the Escherichia coli ClC channel structure as a template. This model agrees well with AE1 cysteine scanning mutagenesis data and blood group antigen sites. To investigate the transport mechanism of AE1, point mutations were introduced in regions of the AE1 homology model corresponding to sites involved in the ClC transport mechanism. The transport activity of these mutants was assessed by Cl-/HCO3- exchange assays in HEK293 cells and Xenopus laevis oocytes. Several AE1 mutations, at sites corresponding to ClC transport mechanism residues, resulted in significant changes in transport activity compared to wild-type AE1, without changes in electrogenicity or transport stoichiometry. A study of the E. coli ClC dimer interface identified tryptophan mutations, which disrupted the dimer interface of ClC. In a similar fashion, AE1 tryptophan mutations were made in an AE1 membrane domain background, using the AE1 homology model as a guide. The majority of AE1 tryptophan mutations decreased AE1 protein expression; however, no disruptions of the dimer interface were observed using chemical crosslinking. Chemical crosslinking may not have the sensitivity to monitor slight disruptions in the AE1 dimer interface, and thus alternate strategies to monitor the oligomeric state of AE1 tryptophan mutants must be investigated. Together, these studies have provided valuable insights into the structure and transport mechanism of AE1.
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
P.T. Bonar, J.R. Casey, Plasma membrane Cl-/HCO3- exchangers: Structure, mechanism and physiology. Channels (Austin) 2, 337-345 (2008).P. Bonar, J.R. Casey, Purification of functional human Cl-/HCO3- exchanger, AE1, over-expressed in Saccharomyces cerevisiae. Protein Expr Purif. 74, 106-115 (2010).P.T. Bonar, H.P. Scheinder, H.M. Becker, J.W. Deitmer, and J.R. Casey, Three-Dimensional Model for the Human Cl-/HCO3- exchanger, AE1, by Homology to the E. coli ClC Protein. Journal of Molecular Biology. (2013).

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