The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane chloride channel expressed in epithelial tissues. While protein kinase A (PKA) is the main activator of CFTR, patch clamp data have suggested a permissive role for PKC in modulating PKA responses. To clarify the role of PKC in CFTR regulation we used PCR mutagenesis to construct a CFTR mutant (9CA) in which the serines or threonines in nine consensus sequences for PKC phosphorylation were replaced by alanines. Its expression in stably transfected baby hamster kidney (BHK) cells was consistently lower than that of wild-type (WT) CFTR. Nevertheless, 9CA cells displayed a robust cAMP-stimulated iodide efflux which was comparable in magnitude to that of WT CFTR cells when normalised for protein expression level. The cpt-cAMP concentration-response relationship in intact cells was similar for WT and 9CA CFTR (EC50 ≈ 100muM), however the onset of iodide efflux from cells expressing the mutant was markedly delayed at all cpt-cAMP concentrations tested. This delay was mimicked by pre-treating WT cells with the PKC inhibitor chelerythrine (10muM), and chelerythrine did not cause further slowing of iodide efflux from 9CA cells. In vitro phosphorylation by PKA and PKC was similar for WT and 9CA CFTR and phosphorylation in the presence of both kinases was additive. / A major impediment to understanding CFTR structure/function has been the difficulty of obtaining sufficient purified CFTR protein for biochemical studies. In an attempt to overcome this problem we expressed a His-tagged CFTR construct in the methylotrophic yeast Pichia pastoris. The final yield of CFTRHis10 following solubilization of Pichia membranes in 0.5% lysophosphatidylglycerol (LPG) and nickel chelate chromatography was ∼20mug/litre. CFTRHis10 was not glycosylated in this yeast system. Both PKA and PKC phosphorylated semi-purified CFTRHis10 in vitro and phosphorylation in the presence of both kinases was additive.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.33407 |
| Date | January 2000 |
| Creators | Hinkson, Deborah Anne Rochelle. |
| Contributors | Hanrahan, John W. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Physiology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001771625, proquestno: MQ70715, Theses scanned by UMI/ProQuest. |
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