Plasma membrane Na+/H+ exchangers (NHEs) have been shown to be inhibited by amiloride and its derivatives. Studies have demonstrated that these antagonists act on the transporter by interacting with amino acid residues within certain transmembrane domains (TM4 and 9). Experiments in our laboratory have identified three novel sites involved in inhibitor interactions (PP157--8, E350, and G356). The general focus of this thesis was to study the structure-function relationship of NHE1. One of the aims of this thesis was to assess whether mutations at these novel sites also affected transport kinetics (i.e., Na+ and H+ affinity). The results showed that neither Na+ nor H+ affinities were significantly altered with any of the mutations analyzed. The next objective was to investigate the nature of the interaction between the exchanger and pharmacological agents, by measuring transport activity in the presence of substituted guanidinium antagonists. The results suggest an interaction between L167 and the chlorine moiety at position 3 of the benzoyl group of a novel benzoyl guanidinium compound, while G356 appears to interact with the chlorine moiety at position 4 (rather than position 3) of the benzene ring. / The last objective was to define the membrane topology of NHE1. The methodology involves reintroducing cysteine residues into a cysteine-less mutant NHE1 and assessing their accessibility with thiol-reactive agents. Unfortunately, these results were inconclusive and further optimization of the assay conditions is required.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.33410 |
| Date | January 2000 |
| Creators | Iannuzzi, Pietro. |
| Contributors | Orlowski, John (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Physiology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001771685, proquestno: MQ70718, Theses scanned by UMI/ProQuest. |
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