Inhibition of ATP-regulated K+ channels precedes depolarization-induced increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells.

Establishment and Application of in Vitro Membrane Potential Assays in Cell Lines with Endogenous or Recombinant Expression of ATP-Sensitive Potassium Channels (Kir6.2/SUR1) Using a Fluorescent Probe Kit

“…The individual cells stained uniformly in the cytoplasm, whereas the nuclei did not seem to pick up fluorophore (data not shown). The staining patterns were very similar to what has been reported for DiBAC 4 (3)another fluorescent probe for qualitative membrane potential measurements that we have used for screening purposes in βTC3 cells 15 -and more biologically oriented work in an identical islet preparation, 17 so we consider it highly likely that the new fluorophore behaves in a similar fashion as DiBAC 4 (3). A single depolarization experiment using 30 mM KCl in a pancreatic islet also suggested an increase in fluorophore uptake during depolarization without a change in the distribution patterns (data not shown).…”

“…The present membrane potential kit for FLIPR™ comprises an additional technique-extracellular dye quenching-to further reduce contribution from extracellular fluorophore, and we found it interesting to investigate if this new feature would make it possible to go back to a conventional fluorescence plate reader for easier and cheaper compound testing and profiling with increased capacity. We hereby demonstrate that this is indeed the case, and we find that the new assay in βTC3 cells is more sensitive and stable than our old protocol based on DiBAC 4 (3). The insulin-producing lines, such as βTC3, have the clear advantage that their glucose metabolism can be used to maintain a state of metabolic channel closure, but they have the disadvantage of being of rodent origin and having a very long doubling time.…”

“…15,17 Due to high background fluorescence from extracellular dye, this assay cannot run in a conventional fluorescence plate reader. In fact, the FLIPR™, which optically reduces the background fluorescence, was developed originally for microplate-based assays using DiBAC 4 (3). The present membrane potential kit for FLIPR™ comprises an additional technique-extracellular dye quenching-to further reduce contribution from extracellular fluorophore, and we found it interesting to investigate if this new feature would make it possible to go back to a conventional fluorescence plate reader for easier and cheaper compound testing and profiling with increased capacity.…”

“…Thus, an increase in extracellular glucose reduces the open probability of the K ATP channels by increasing the ATP/ADP ratio. 2,3 This is, at glucose levels above basal, followed by depolarization, Ca 2+ influx through opening of the voltage-dependent Ca 2+ channels (VDCCs) of the L type, and exocytosis of insulin-containing granules. 4 When the glucose levels, through the action of insulin released into the bloodstream, return to basal, the ATP/ADP ratio decreases and the K ATP channels open, repolarizing the plasma membrane, closing the VDCCs, and effectively returning the βcells to the resting state.…”

“…4 When the glucose levels, through the action of insulin released into the bloodstream, return to basal, the ATP/ADP ratio decreases and the K ATP channels open, repolarizing the plasma membrane, closing the VDCCs, and effectively returning the βcells to the resting state. 3 Besides the presence and important function in the pancreatic β-cells, the Kir6.2/SUR1 channel isoform has been reported to be present in glucagon-secreting α-cells, [5][6][7] GLP-1-containing intestinal L-cells, 8 and glucose-responsive neurons in the hypothalamus, 9 where its potential importance is yet to be determined. All in all, this makes Kir6.2/SUR1 into an interesting drug target.…”