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

• Towards a Structural Model of AE1

• Author / Creator

  Bonar, Pamela T

• 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.

• Subjects / Keywords

  • Homology model
  • Protein expression and purification
  • AE1 physiology
  • AE1 structure
  • Bicarbonate transport
  • Dimer interface

• Graduation date

  Fall 2013

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3N095

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.