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dc.contributor.advisorHamilton, Kirk L
dc.contributor.authorFarquhar, Rachel
dc.date.available2015-03-24T03:15:02Z
dc.date.copyright2015
dc.identifier.citationFarquhar, R. (2015). The role of cytoskeletal elements in the trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/5567en
dc.identifier.urihttp://hdl.handle.net/10523/5567
dc.description.abstractThe intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane in polarized epithelia where it plays an essential role in promoting trans-epithelial ion transport. KCa3.1 is found in many tissues in the body and plays an important role in many physiological and pathological processes (e.g., regulation of salt and fluid transport in the gastrointestinal tract, atherosclerosis, sickle cell disease and asthma). Functional KCa3.1 must be targeted to the basolateral membrane, a process that is dependent upon proper cytoskeletal function. The cytoskeleton is comprised of actin and microtubule filaments. Actin filaments are comprised of polymerised G-actin monomers bound to form filamentous F-actin strands. Microtubules are long filamentous structures comprised of tubulin subunits, made from α-tubulin and β-tubulin monomers. This study examines the role of microfilaments and microtubules in the trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells. To address this, Fischer Rat Thyroid cells grown on filter inserts to form a confluent epithelium were stably transfected with the Biotin Ligase Acceptor Peptide (BLAP)-KCa3.1 construct. This construct allowed for the selective labeling of basolaterally expressed KCa3.1 using streptavidin. Selective labeling of membrane bound KCa3.1 allowed for the measurement of changes in KCa3.1 expression, in response to drugs that disrupt cytoskeletal elements, to reflect changes in KCa3.1 located on the basolateral membrane. This measure allowed for a direct correlation to be drawn between targeted disruption of specific cytoskeletal elements, e.g. microtubules and microfilaments, and expression of basolaterally-located KCa3.1. PCR was used to determine the mRNA expression levels of KCa3.1 in stably transfected cell lines and SDS-PAGE techniques were employed to investigate protein expression levels of KCa3.1. Western blotting was used to explore the effects of Cytochalasin D (Cyto D), Latrunculin A (Lat A), and Myosin Light Chain Inhibitor-7 (ML-7) which inhibit the function of actin (Cyto D, Lat A) and myosin light chain kinase (ML-7) respectively. Toxicity tests were performed to determine cell survival under a range concentrations of 0-20 μM (0, 3, 5 hr) for all three drugs with cell survival reduced with 20 μM at t = 5 hr for Cyto D and Lat A. Cyto D was administered over intervals of 0, 3 and 5 hr at 10 μM resulting in a decreased relative expression of KCa3.1 (compared to control) of 0.6±0.14 at t = 3 and further decrease in the expression of the channel at t = 5 hr with a relative expression of 0.12±0.035 (n = 5, p < 0.05). Lat A was also administered over intervals of 0, 3 and 5 hr at 10 μM causing a relative reduction in the expression of KCa3.1 at the basolateral membrane compared to the control. At t = 3 hr the expression of KCa3.1 was reduced to 0.7±0.065 and decreased to 0.3±0.049 at t = 5 hr (n = 4, p < 0.001). Finally, cells treated with microtubule inhibitor ML-7 showed a relative reduction in KCa3.1 expression of 0.55±0.12 at t = 3 hr, the expression was further decreased to 0.33±0.11 at t = 5 hr compared to the control. These data confirm that microtubules and microfilaments of the cytoskeleton are crucial in trafficking KCa3.1 to the basolateral membrane of polarised epithelial cells.
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.subjectactin
dc.subjectmyosin
dc.subjectmicrotubules
dc.subjectcytoskeleton
dc.subjectwestern blot
dc.subjectpolymerase chain reaction
dc.subjectPCR
dc.subjectbiotinylation
dc.subjectbiotin
dc.subjecttoxicity test
dc.subjectCytochalasin D
dc.subjectMyosin Light Chain 7
dc.subjectLatrunculin A
dc.subjectCyto D
dc.subjectML-7
dc.subjectFischer Rat Thyroid
dc.subjectFRT
dc.subjectKCa3.1
dc.subjectKCNN4
dc.subjectBasolateral membrane
dc.subjectepithelial
dc.subjectcell culture
dc.subjectmembrane expression
dc.subjectlabeling
dc.subjectBLAP
dc.subjectbiotin ligase acceptor peptide
dc.subjectRachel Farquhar
dc.subjectFarquhar
dc.subjectLat A
dc.subjectSK4
dc.subjectIK1
dc.subjectK+
dc.subjection channel
dc.subjectcalcium activated
dc.subjectinward rectifying
dc.subjectintermediate conductance
dc.subjectcell proliferation
dc.subjectcell migration
dc.subjectfluid regulation
dc.subjecttox test
dc.titleThe role of cytoskeletal elements in the trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells
dc.typeThesis
dc.date.updated2015-03-24T02:40:31Z
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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