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 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 beta-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 alpha-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.
Arkhammar
Per
P
Discovery, Novo Nordisk A/S, Måløv, Denmark. poga@novonordisk.com
Wahl
Philip
P
Gerlach
Berit
B
Fremming
Tinna
T
Hansen
John B
JB
D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007328:Insulin / Q000096:biosynthesis; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D011786:Quality Control; D011994:Recombinant Proteins / Q000378:metabolism; D000099161:Kcnj11 Channel
D005456:Fluorescent Dyes; D007328:Insulin; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D000099161:Kcnj11 Channel