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Title: Single-Channel Characterisation of Potassium Channels with High Temperature Studies.
Author: Mason, Amy
ISNI:       0000 0001 3620 6535
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2008
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Potassium channels control the conduction of K+ across cell membranes, down their electrochemical gradient. This rapid and highly selective movement of K+ is essential to many biological processes. K+ channels are largely alpha helical, tetrameric proteins that span the lipid bilayer. Diversity in K+ channels arises primarily in the mechanism of gating by various ligands or voltage; however, the basic structural elements, notably the selectivity filter are conserved within the family. Studies ~n this thesis focus on the single-channel behaviour of the K+ channels KcsA and Kcv. KcsA is a proton-activated channel from the bacterium Streptomyces lividans and its crystal structure was the first of a K+ channel to be solved. Kcv expressed by the Paramecium bursaria Chlorella virus is the smallest known K+ channel and thus represents the minimal structural entity necessary to form a functional and selective pore. Studies on the bacterial inward rectifying channels, KirBacs, have also been initiated. The KirBacs are a superfamily of prokaryotic channels homologous to eukaryotic Kir channels. In this work, KcsA and Kcv were found to form stable tetramers, which can be expressed by coupled in vitro transcription and translation and purified by polyacrylamide gel electrophoresis. The purified tetramers were reconstituted into planar lipid bilayers and studied at the single-channel level. Through single-channel recordings the ionic selectivity, gating behaviour, functional effects of site-directed mutagenesis and the interaction between Kcv and blockers have been studied. In addition, single-channel studies at elevated temperatures have revealed the remarkable thermostability of Kcv, as well as insight into the transport of ions through a narrow and selective pore. Through temperature studies, it has been possible to obtain the thermodynamic and kinetic parameters describing Kcv activity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available