Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.

ATP-sensitive potassium (K ATP ) channels are abundantly expressed in the myocardium.Although a definitive role for the channel remains elusive they have been implicated in the phenomenon of cardioprotection, but the precise mechanism is unclear. We set out to test the hypothesis that the channel protects by opening early during ischemia to shorten action potential duration and reduce electrical excitability thus sparing intracellular ATP. This could reduce reperfusion injury by improving calcium homeostasis.Using a combination of contractile function analysis, calcium fluorescence imaging and patch clamp electrophysiology in cardiomyocytes isolated from adult male Wistar rats, we demonstrated that the opening of sarcolemmal K ATP channels was markedly delayed after cardioprotective treatments; ischemic preconditioning, adenosine and PMA. This was due to the preservation of intracellular ATP for longer during simulated ischemia therefore maintaining sarcolemmal K ATP channels in the closed state for longer. As the simulated ischemia progressed, K ATP channels opened to cause contractile, calcium transient and action potential failure, however there was no indication of any channel activity early during simulated ischemia to impart an energy sparing hyperpolarization or action potential shortening.We present compelling evidence to demonstrate that early opening of sarcolemmal K ATP channels during simulated ischemia is not part of the protective mechanism imparted by ischemic preconditioning or other PKC-dependent cardioprotective stimuli. On the contrary, channel opening was actually delayed. We conclude that sarcolemmal K ATP channel opening is a consequence of ATP depletion, not a primary mechanism of ATP preservation in these cells. 3Highlights:• Opening of the SarcoK ATP channel was proposed to be cardioprotective• Channel opening was delayed after cardioprotective stimuli• Ca 2+ & ATP levels were maintained during ischemia independent of SarcoK ATP opening• Mitochondrial function preserved during ischemia, independent of SarcoK ATP opening• Early opening of SarcoK ATP is not involved in PKC-dependent cardioprotection

C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K + channels (K ATP ). To investigate the protective role of K ATP channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294-1358, the A-fragment, reduced sarcolemmal K ATP currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 ± 1.22; and A-fragment 0.94 ± 0.07 pA pF −1 , n = 6,6, P < 0.001). An inactive fragment (1358-1545, current density 6.30 ± 0.85 pA pF −1 , n = 6 ) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca 2+ loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca 2+ and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca 2+ loading, was abolished by the A-fragment. Our data are consistent with a role for K ATP channels in causing action potential failure and reduced Ca 2+ loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K ATP channel, but we also discuss the possibility of mitochondrial effects.

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