The Role of Sulphonylurea Receptor Splice-Variants in Ischemic Preconditioning and Cardioprotection

ATP-sensitive potassium channels (KATP) play a vital role in important physiological processes, such as insulin secretion by the pancreas and the ability of heart muscle to withstand periods of ischemia (cardioprotection). KATP channels are hetero-octamers of 4 pore-forming Kir6 subunits and 4 sulphonylurea receptor (SUR) subunits. SUR subunits, which are essential for channel expression, modulate the activity of the channel pore, making it sensitive to the stimulatory effects of nucleotides and are the target of various pharmacological agents (including the anti-diabetic sulphonylureas). Sulphonylurea receptor subunits are encoded by two genes, SUR1 and SUR2; which has two major (SUR2A and SUR2B), and several minor, splice-variants.
Gene knock out has, so far, failed to definitively identify the SUR isoforms responsible for cardioprotection or the mechanisms involved. Over-expression studies have also produced contradictory results. A second unknown is the cellular compartment in which the channel is having its beneficial effects; some evidence favours a sarcolemmal location and some a mitochondrial one. Increases in sarcoKATP channel number have been associated with ischemic preconditioning (IPC, the protection offered by brief episodes of ischemia prior to a prolonged period) and it has previously been reported that PMA treatment (which stimulates IPC) leads to a doubling of the Kir6.2 content of mitochondria. Deliberate targeting of Kir6.2 to mitochondria has also been shown to increase tolerance of ischemia. These sub-cellular pools are unlikely to be static and channel number seems to be subject to dynamic regulation through internalisation, re-cycling and possibly exchange between different compartments.

The project will explore the hypothesis that specific SUR2 splice-variants are responsible for IPC and cardioprotection; that they are preferentially trafficked to specific intracellular compartments; and that stimuli which trigger IPC lead to a redistribution of KATP channels. We have developed a series of adenoviruses expressing short hairpin RNAs targeting different KATP channel subunits including the SUR2 splice-variants. We will complement this approach by comparing the effects of controlled expression of specific splice-variants in ventricular myocytes (using drug-inducible promoters). We will also investigate the sub-cellular distribution of SUR2 splice-variants by confocal microscopy and fluorescence resonance energy transfer (FRET) and track their movement in response to pre-conditioning stimuli.


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