KATP channels are ubiquitously expressed and link metabolic state to electrical excitability. In heart, in response to ischaemic stress, they play a protective role and in vascular smooth muscle regulation of vascular tone (vasorelaxation). Functional KATP channels are hetero-octamers composed of two subunits, a pore forming Kir6, which is a member of the inwardly rectifying potassium channels family and a regulatory sulphonylurea receptor (SUR). In response to nucleotides and pharmacological agents, SUR allosterically regulate KATP channel gating. Multidisciplinary techniques (molecular biology, biochemistry, electrophysiology, pharmacology) were used to study the allosteric regulation between these two heterologous subunits in KATP channels. This project was divided into three major sub-projects: 1) Application of site directed mutagenesis and biochemical techniques to identify the cognate interaction domain on Kir6.2 for SUR2A-NBD2 (nucleotide binding domain 2). 2) Electrophysiological techniques to investigate the allosteric information transfer between heterologous subunits Kir6 and SUR2A. 3) Recombinant fusion protein to express and purify the cytoplasmic domains of Kir6.2 for structural analysis of the interaction between the two subunits. This study reports on the identification of three cytoplasmic electrostatic interfaces between Kir6 and SUR2A involved in determining the sensitivity of KATP channel agonist, pinacidil, and antagonist, glibenclamide, from SUR2A to the Kir6 channel pore. For structural study of cytoplasmic domains of Kir6.2, bacterial TM1070 was used as fusion partner with Kir6.2. A TM1070-Kir6.2 NC (CT-His6 tag) fusion construct expressed in Arctic Express competent cells permitted successful expression of folded cytoplasmic domains of Kir6.2 in near native form. Immobilized metal ion affinity chromatography, IMAC (Ni2+), and gel filtration chromatography (GFC) column as second purification step were performed to purify this recombinant protein. The purification was confirmed by CBS and Western blot analysis. Possibly, this new information on channel structure-function relationships may contribute to the design of novel and more effective drugs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:556298 |
| Date | January 2012 |
| Creators | Rubaiy, Hussein Nori |
| Contributors | Norman, Robert. : Lodwick, David |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/11003 |
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