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dc.contributor.advisorMcDonald, Fiona Jean
dc.contributor.authorGeda, Anna Caterina
dc.date.available2015-11-15T22:27:48Z
dc.date.copyright2015
dc.identifier.citationGeda, A. C. (2015). The role of retromer in the epithelial sodium channel trafficking pathway (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/6076en
dc.identifier.urihttp://hdl.handle.net/10523/6076
dc.description.abstractThe epithelial sodium channel (ENaC) is a protein located at the apical membrane of polarised epithelial cells, primarily expressed in the epithelia of the gastrointestinal tract, lungs and kidney. ENaC's main function is that of absorbing sodium and it is strongly involved in regulating and maintaining total-body salt and water homeostasis, acting as the rate-limiting step for sodium reabsorption into the body. Its activity, therefore, is crucial for determining blood volume and, as a consequence, blood pressure. The sorting and trafficking of ENaC to the apical membrane is a tightly controlled process, requiring the interaction of multiple proteins and organelles. Although ENaC has been well-characterised, there are certain aspects about its trafficking which need to be clarified, such as defining the many proteins involved in the recycling of the channel to and from the apical membrane. A potential, novel candidate involved in ENaC recycling is the retromer complex. This endosome-associated protein complex has been shown to have a role in protein recycling, as well as maintaining cell polarity by assisting in the transport of proteins to and from their appropriate membrane. The aim of this study was to investigate whether retromer is involved in the recycling of ENaC in polarised epithelia, focusing on three specific proteins, namely ccdc22, Snx4 and KIBRA. Whilst ccdc22 is an established component of the retromer complex, Snx4 and KIBRA were hypothesised to be part of retromer, a plausible concept given their cellular localisation and proposed function. To test whether ccdc22, Snx4 and KIBRA were involved in ENaC recycling, their function was altered (via protein knockdown or overexpression) and the effects on ENaC trafficking were measured. Using transiently transfected HEK293 (human embryonic kidney) and FRT (Fischer rat thyroid) cells, semi-quantitative analysis was carried out with Western blots to visualise whether the knockdowns/overexpression of the proteins of interest were occurring. Then, Ussing chamber experiments were conducted to detect any changes in the ENaC channel’s activity at the apical membrane when a retromer protein was knocked down or over-expressed. Finally, GST-pulldown assays were performed to visualise whether the ENaC channel interacted with retromer through the protein KIBRA. Significant knockdowns were obtained of both Snx4 (p<0.01, n=5) and ccdc22 (p<0.001, n=5) in FRT cells. Interestingly, the functional data showed that knocking down ccdc22 had no effect on the activity of ENaC at the apical membrane (n=6), whilst knockdown of Snx4 (n=7) led to a significant increase (p<0.001) in the amiloride-sensitive short circuit current (Isc). Furthermore, overexpression of the KIBRA protein caused a significant decrease in the amiloride-sensitive Isc (p<0.001, n=5). Finally, the in vitro GST-pulldown assay preliminarily showed an interaction between KIBRA and the βENaC subunit, possibly suggesting that ENaC and retromer interact. Overall, this research project represents an important initial step towards clarifying the mechanism of ENaC recycling to and from the apical membrane of epithelial cells and as a result, the regulation of ENaC channels present at the cell membrane. Understanding how ENaC recycling occurs is essential, given the important role this channel has to play in maintaining the body's blood pressure.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectENaC
dc.subjectretromer
dc.subjectprotein trafficking
dc.subjectKIBRA
dc.subjectSnx4
dc.subjectccdc22
dc.subjectUssing chamber
dc.subjectprotein recycling
dc.subjectendocytosis
dc.subjectepithelial sodium channel
dc.titleThe role of retromer in the epithelial sodium channel trafficking pathway
dc.typeThesis
dc.date.updated2015-11-15T21:37:17Z
dc.language.rfc3066en
thesis.degree.disciplinePhysiology
thesis.degree.nameBachelor of Biomedical Sciences with Honours
thesis.degree.grantorUniversity of Otago
thesis.degree.levelHonours
otago.interloanno
otago.openaccessAbstract Only
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