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A study of cloned and native KATP channels in HEK 293 cells and rat ventricular myocytes.
thesisposted on 2009-04-30, 12:06 authored by Richard Daniel Rainbow
The properties of the cardiac isoform(Kir6.2/SUR2A) of the ATP sensitive potassium (KATP) channel expressed in HEK 293 cells and from isolated rat ventricular myocytes were investigated using a variety of methods. The ATP sensitivity and sulphonylurea drug sensitivity of the channel in both expression systems was investigated using patch clamp techniques in both excised patch and whole cell configurations. The potential for heteromultimerisation between the two pore forming subunits of the KATP channel complex, Kir6.1 and Kir6.2, was investigated by the generation of a Kir6.1 /Kir6.2 fusion protein. Molecular biological techniques were applied to construct the fusion protein and electrophysiology in combination with specific Kir6.1 and Kir6.2 antibody labelling was used to determine successful expression of the fusion protein. The interactions between the structurally unrelated Kir6.2 and SUR2A were probed with the use of protein fragments of SUR2A containing a region of potential interaction identified biochemically. It was found that the fragments of SUR2A containing the potential region of interaction disrupted expression of functional channels at the cell surface as shown by electrophysiological recordings from HEK 293 cells stably expressing Kir6.2/SUR2A and transiently transfected with the protein fragments. Immunohistochemical staining of the channel complex with Kir6.2 and SUR2A specific antibodies was also used and showed a decreased surface expression of KATP channels in a population of cells. The protein fragments were then expressed in rat ventricular myocytes to reduce the number of functional channels at the cell surface. This allowed the role of the KATP channel in the response to metabolic inhibition to be investigated. It was shown that the rat ventricular myocytes transfected with the protein fragments entered a state of contractile failure earlier than their non-transfected counterparts and also a state of rigor contracture earlier. The transfected myocytes ability to recover calcium homeostasis on reperfusion after metabolic inhibition was also compromised.
Supervisor(s)Standen, Nick; Davies, Noel
Date of award2003-01-01
Awarding institutionUniversity of Leicester