Please use this identifier to cite or link to this item:
Title: A comparison of the pore-forming subunits of the ATP-sensitive potassium channel
Authors: Chadburn, Andrew James
Supervisors: Lodwick, Dave
Standen, Nick
Award date: 24-Mar-2010
Presented at: University of Leicester
Abstract: Kir6.1 and Kir6.2 display several key differences in characteristics e.g. Kir6.2 can open spontaneously upon removal of inhibitory nucleotides while Kir6.1 cannot. Residues responsible for some of these disparate properties have been mapped to the N-terminus of Kir6.0. Mutant cDNAs for these subunits were created where amino acid(s) were taken from one isoform and replaced with the corresponding residue(s) from the other. The whole cell and inside-out patch clamp techniques were used to assess the current density, spontaneous opening, ATP sensitivity and unitary conductance of these mutant channels. All mutants showed similar current density to their parent channels, suggesting similar expression. One mutant, which exchanged residues 15-17 of Kir6.2 (TRL) for ARI, had significantly lower conductance than wild type, possibly due to allosteric modulation of the conduction pathway. Simultaneous mutation of a group of residues from Kir6.2 (S37A, K39S, N41A and V44L) reduced relative activation, a measure of spontaneous opening. However, mutation of these residues individually had no effect, suggesting that they interact to affect spontaneous opening in wild type channels. 14-3-3 are a family of chaperone and scaffold proteins shown to interact with several ion channels both functionally and during expression. 14-3-3 ε and ζ have been implicated in expression of KATP channels. To try and address the poor expression of Kir6.1-based channels, 14-3-3 ε and ζ were co-expressed with KATP channels. No effect was seen with any 14-3-3 isoforms co-expressed with Kir6.1. However, 14-3-3 ε significantly increased current density of Kir6.2/SUR2A channels. Co-expression of 14-3-3 ε and ζ attenuated this effect. The disparate effects of different combinations of these 14-3-3 isoforms suggest that their dimerisation may play a role in the effect seen here.
Type: Thesis
Level: Doctoral
Qualification: PhD
Sponsors / Funders: British Heart Foundation
Rights: Copyright © the author, 2010.
Appears in Collections:Theses, Dept. of Cardiovascular Sciences
Leicester Theses

Files in This Item:
File Description SizeFormat 
2009chadburnajphd.pdf2.94 MBAdobe PDFView/Open

Items in LRA are protected by copyright, with all rights reserved, unless otherwise indicated.