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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

Discovery, Novo Nordisk A/S, Måløv, Denmark

Philip Wahl

Discovery, Novo Nordisk A/S, Måløv, Denmark

Berit Gerlach

Discovery, Novo Nordisk A/S, Måløv, Denmark

Tinna Fremming

Discovery, Novo Nordisk A/S, Måløv, Denmark

John B. Hansen

Discovery, Novo Nordisk A/S, Måløv, Denmark

The flow of current through the adenosine triphosphate (ATP)-sensitive potassium channel (K_ATP) of the isoform Kir6.2/SUR1 regulates the resting membrane potential in the pancreatic ß-cell. In combination with the cellular glucose metabolism, it is an important minute-to-minute regulator of insulin secretion and whole-body glucose homeostasis. The same K_ATP isoform is further reported to be present in glucagon-secreting -cells, intestinal L-cells, and glucose-responsive neurons in the hypothalamus. All in all, this makes Kir6.2/SUR1 an interesting drug target. Using a commercially available fluorescent membrane potential probe kit and a conventional 96-well fluorescence plate reader, the authors have developed and established qualitative membrane potential assays used to screen for potassium channel closers (KCCs) and openers (KCOs) in insulin- and glucagon-secreting cell lines as well as in cells with recombinant expression of the human Kir6.2/SUR1 channel complex. Both glucose- and KCC-induced depolarization could be demonstrated. The magnitudes of these responses and KCO-induced repolarization at high glucose displayed some variation between the different cell lines but a similar rank order of test compounds. Some cell types required the presence of a KCC, such as tolbutamide, to display significant effects of KCOs. The authors find that robust and reliable functional in vitro assays compatible with medium-throughput screening and high-throughput screening can be developed as a base for finding new, more potent, and isoform-selective KCCs and KCOs.

Key Words: fluorescent membrane potential probe kit • KATP • Kir6.2/SUR1 • potassium channel openers and blockers • HTS • fluorescence plate reader

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