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dc.contributor.advisorHamilton, Kirk
dc.contributor.advisorMcDonald, Fiona
dc.contributor.authorPilmore, Elliot James
dc.date.available2018-04-06T03:44:16Z
dc.date.copyright2018
dc.identifier.citationPilmore, E. J. (2018). Retromer associated sorting nexins SNX1, SNX4, and SNX27 and the trafficking and basolateral membrane population of the intermediate conductance calcium activated potassium channel (KCa3.1) in polarised epithelial cells (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/7995en
dc.identifier.urihttp://hdl.handle.net/10523/7995
dc.description.abstractThe intermediate conductance calcium (Ca 2+)-activated potassium (K+) channel (KCa3.1) is targeted to the basolateral membrane of polarised epithelial cells, and is also found in many nonepithelial cell types. KCa3.1 performs several fundamental roles, including the promotion of transepithelial ion transport, and the maintenance of a homeostatic equilibrium inside cells. Thus, KCa3.1 is found in a wide range of tissues, from epithelial to neural, and is involved in several disease states. These disease states include sickle-cell anaemia, cardiac fibrosis, and diabetic nephropathy, and paint KCa3.1 as a potential target for novel disease therapies. In order to create these novel disease therapies, it is vital to understand how KCa3.1 is trafficked within the cell. Functionally, KCa3.1 is trafficked from the ER to the Golgi in a manner dependent on Rab1, and from the Golgi to the plasma membrane in a manner dependent on Rab8. Additionally, KCa3.1 trafficking has been shown to require a functional cytoskeleton, as well as the motor protein Myocin-Vc. One trafficking pathway which may traffic KCa3.1 is the Retromer pathway. This pathway involves multiple protein complexes, including the cargo recognition complex, and the WASH complex, as well as multiple individual sorting nexin proteins. If KCa3.1 is found to interact with components of the Retromer pathway, it could lead to new disease therapies. This project examined the role of three distinct sorting nexin protein; SNX1, SNX4, and SNX27, on the trafficking and basolateral membrane population of KCa3.1 in polarised epithelial cells. In order to do this, Fischer Rat Thyroid cells transfected to stably express a KCa3.1-BLAP (biotin ligase acceptor peptide) construct, were transfected with SNX1, 4, or 27 specific siRNA, then grown on filters in order to achieve polarity. This allowed for the apical and basolateral membrane populations of KCa3.1 to be independently assessed. Immunoblots were used to determine the extent to which transfections were successful. In cells where the transfection was successful, the basolateral membrane population of KCa3.1 was determined by immunoblots. Additionally, Ussing chamber experiments were utilised in order to explore the effects of transfections on the KCa3.1 current when stimulated by the KCa3.1 opener 1-EBIO, or the KCa3.1 inhibitor clotrimazole. This project showed the first evidence of the endogenous expression of SNX1 and SNX27 in FRT cells, and confirmed expression of SNX4 in FRT cells, first discovered in the McDonald Lab. Following this demonstration, cells were transfected with 40 pM of either SNX1, SNX4, or SNX27 siRNA. The transfection with SNX1 siRNA resulted in a 44 ± 8% decrease in SNX1 protein levels, however, showed no significant decrease in the basolateral membrane population of KCa3.1, nor in the KCa3.1 sensitive current measured by Ussing chamber experiments was observed. The transfection with SNX4 showed a 62 ± 12% decrease in intracellular SNX4, however, similar to SNX1, a decrease in intracellular SNX4 did not appear to affect either the basolateral membrane population of KCa3.1, nor the KCa3.1 sensitive currents. Finally, the transfection of cells with SNX27 siRNA showed a 27 ± 4% decrease in the intracellular protein levels of SNX27. While knockdown of SNX27 did not elicit a significant change in the basolateral membrane population of KCa3.1, it did suggest the possibility of KCa3.1 missorting to the apical membrane, as there was a trend towards a significant increase in the apical membrane population of KCa3.1 in the SNX27 knockdown cells compared to the control cells (p = 0.0818; n=4). These results appear to be consistent with the notion that KCa3.1 is not recycled in polarised epithelial cells, and suggest that KCa3.1 is not trafficked via the Retromer pathway. While KCa3.1 does not appear to be trafficked via the Retromer pathway, there are still unknown factors regarding the trafficking of KCa3.1 which need to be explored, such as why KCa3.1 appears to be recycled in migratory cells. It is important to fully comprehend the trafficking mechanisms surrounding KCa3.1 in order to develop novel effective therapeutic techniques to combat pathophysiological conditions resulting from dysfunctional KCa3.1.
dc.format.mimetypeapplication/pdf
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.subjectKCa3.1
dc.subjectCalcium
dc.subjectPotassium
dc.subject"Calcium activated
dc.subjectCalcium-activated
dc.subjectCalcium activated potassium channel
dc.subjectCalcium-activated potassium channel
dc.subjectSNX
dc.subjectSorting nexin
dc.subjectRetromer
dc.subjectTrafficking
dc.subjectEpithelial
dc.subjectFischer Rat
dc.subjectSNX1
dc.subjectSNX4
dc.subjectSNX27
dc.subject1-EBIO
dc.subjectClotrimazole
dc.subjectBLAP
dc.titleRetromer associated sorting nexins SNX1, SNX4, and SNX27 and the trafficking and basolateral membrane population of the intermediate conductance calcium activated potassium channel (KCa3.1) in polarised epithelial cells
dc.typeThesis
dc.date.updated2018-04-06T03:02:59Z
dc.language.rfc3066en
thesis.degree.disciplinePhysiology
thesis.degree.nameMaster of Science
thesis.degree.grantorUniversity of Otago
thesis.degree.levelMasters
otago.openaccessOpen
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