Regulation of KATP channel activity

Thesis

ATP-sensitive potassium (KATP) channels are present in many tissues, most notably pancreatic islets and cardiac cells, where they couple the metabolic state of a cell to its electrical activity by regulating the flow of K+ across the membrane in response to the intracellular ATP/ADP ratio. KATP channels are an octameric complex, comprised of four inwardly-rectifying potassium channel (Kir) subunits, each of which is associated with a sulphonylurea receptor (SUR) subunit. In pancreatic islets, KATP channels are formed by Kir6.2 and SUR1. The physiological regulation of KATP activity by the ATP/ADP ratio is the summed contribution of activation by Mg-nucleotides binding to SUR1, and inhibition by nucleotides binding to Kir6.2. Mutations in either Kir6.2 and SUR1 which lead to diseases of insulin secretion are frequently observed to disrupt the nucleotide regulation of the channel. In this thesis, I describe the development and application of a method to directly measure nucleotide binding to Kir6.2, using a fluorescent congener for ATP and Forster resonance energy transfer (FRET). Measuring binding simultaneously with measurements of KATP channel current inhibition confirms that a Monod-Wyman-Changeaux (MWC) type allosteric model is able to describe KATP channel inhibition by nucleotides, and that a single bound nucleotide is enough to close the majority of channels. The combination of binding and current measurements with MWC modelling is then applied to determine how mutations at different residues of Kir6.2 and SUR1 contribute to nucleotide binding and transduction at the inhibitory binding site.

Authors: Usher, S

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Ion channels; Electrophysiology; Non-canonical amino acids; Fluorescence spectroscopy
• Subjects: Metabolism; Ion channels; Fluorescence spectroscopy