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The regulation of the cystic fibrosis transmembrane conductance regulator in human respiratory epithelia

England, Alice (2013) The regulation of the cystic fibrosis transmembrane conductance regulator in human respiratory epithelia. PhD thesis, University of Sheffield.

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Cystic fibrosis transmembrane conductance regulator (CFTR) is activated by cAMP-dependent phosphorylation, and functions as an ATP-dependent chloride channel involved predominantly in the movement of chloride ions to maintain cellular ionic homeostasis. Mutations in this channel cause cystic fibrosis, the most common lethal autosomal recessive disease in caucasians. The regulation of CFTR forms a large body of research; this thesis investigated the role of three potential components of the regulatory machinery – nucleoside diphosphate kinase B (NDPK-B), tyrosine phosphorylation and G proteins. This thesis aimed to: 1. Describe the functional relationship between NDPK-B and CFTR. 2. Describe the effect of altered tyrosine phosphorylation in 16HBE14o- cells on CFTR function. 3. Identify the tyrosine phosphatases involved in CFTR regulation. 4. Explain how alterations in tyrosine phosphorylation change CFTR function. 5. Describe the effect of G protein stimulation on CFTR channels which have already been activated by an increase in cellular cAMP. The whole cell patch clamp technique was used to examine ion channel function in two cell types - human bronchial epithelial cells (16HBE14o-) and baby hamster kidney cells (BHK-21). Due to alterations in the function of cultured cells, it was not possible to describe the functional relationship between the histidine kinase NDPK-B and CFTR. Further work is required in this area to elucidate the role of this protein in CFTR regulation. The use of general tyrosine phosphatase inhibitors resulted in a significant decrease in the CFTRinh-172-sensitive conductance in 16HBE14o- cells, and specific inhibitors ruled out the involvement of PTP1B and Shp1/2 phosphatases. Further work is required to explain how tyrosine phosphatase inhibition alters CFTR function. Finally, G protein stimulation in 16HBE14o- after increasing intracellular cAMP had no significant effect on channel function. This suggested that the cAMP-dependent activation of the channel is the predominant mechanism for stimulating channel function.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield)
Identification Number/EthosID: uk.bl.ethos.570166
Depositing User: Mrs Alice England
Date Deposited: 19 Apr 2013 15:07
Last Modified: 27 Apr 2016 14:12
URI: http://etheses.whiterose.ac.uk/id/eprint/3788

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