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Title: Regulation of Na-K-2CI cotransport by protein phosphorylation and protein-protein interactions
Author: Hegney, Karen Louise
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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The first aim of this thesis was to study the phosphorylation of NKCC found in ferret erythrocytes. Immunoprecipitation and subsequent western blotting using total phosphothreonine showed low levels of phosphorylation even when cotransport was not stimulated. This was reduced by ~40% when cotransport was inhibited by the use of kinase inhibitors or Mg2+ removal. When cells were stimulated, by calyculin A or deoxygenation, a doubling of phosphorylation was seen above untreated level. Since the start of this project, the cotransporter kinases, SPAK and OSR, have been discovered. I present western blot evidence that ferret erythrocytes express SPAK and phosphorylated SPAK and may be responsible for NKCC phosphorylation under these conditions. Another aim was to isolate the differently phosphorylated forms of NKCC. A proteomic approach was used, including standard 2-dimensional (2-D) gel electrophoresis. Improvements were made in the overall appearance of 2-D gels. However, this did not translate into improved resolution of membrane proteins in general and more specifically, the cotransporter. Other 2-D electrophoretic methods were employed, with greater success, to look at proteins that interact with NKCC. Blue-native PAGE allows separation of proteins in complex with others and was used to isolate NKCC. The cotransporter was found to separate into two major complexes with apparent molecular masses of 200 and 650 kDa. Mass spectrometric methods identified the erythrocyte anion exchanger, Band 3, in complex with NKCC. Using western blot and PCR techniques, this thesis provides evidence to suggest that not only is NKCC1 present in red blood cells, but that NKCC2 may also be present. This would have implications for the study of cotransport regulation in these cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available