An NMR-based approach to the structural and functional investigation of the Na+/H+ exchanger NHE1

  • Author / Creator
    Lee, Brian L.
  • The Na+/H+ exchanger isoform 1 (NHE1) is the predominant isoform in mammalian cells, and regulates intracellular pH and ion concentrations. NHE1 also interacts with numerous proteins and signalling pathways. Consequently, it has been found to influence cell volume, growth, differentiation, and motility, and has roles in heart disease and cancer. While a wealth of biochemical and physiological data is available on NHE1, little is known about its structure or mechanism of function. In this thesis, a "divide and conquer" approach was used to study the structure and function of NHE1. The structures of individual transmembrane (TM) segments were determined using nuclear magnetic resonance (NMR) spectroscopy, and the function of the TM segments in the full protein were investigated using site-directed mutagenesis in cultured cells. We first examined the structures and functions of the second (EL 2) and fourth (EL 4) extracellular loops of NHE1. Both loops contained functionally important residues, however, EL 2 was found to be structured by NMR while EL 4 was unstructured. Next, we investigated two critical TM segments in NHE1, TM VI and TM XI, as well as TM IV of sod2, a yeast Na+/H+ exchanger. These TM segments were found to have unusual structures consisting of a N- and C-terminal alpha-helix, with an extended segment in between, and the structures correlated well with the functional data. We also looked at larger regions of NHE1 using NMR to examine the TM--TM interactions in the protein, starting with a structure of a two-TM segment of NHE1, TM VI--VII. We also present preliminary NMR experiments on two three-TM segments, TM V--VII and TM X--XII, as well as full-length Escherichia coli NhaA. Overall, the "divide and conquer" approach has allowed us to successfully examine the structures and functions of single-TM segments of NHE1. Furthermore, studies on multi-TM segments and NhaA suggest that we may be able to assemble the structure of NHE1 from its segments or even study the complete protein by NMR spectroscopy.

  • Subjects / Keywords
  • Graduation date
    Fall 2013
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • 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.