Changes in cytoplasmic ATP concentration parallels changes in ATP-regulated K+-channel activity in insulin-secreting cells

Changes in cytoplasmic ATP concentration were monitored in intact insulin-p roducing cells and correlated to changes in the activity of ATP-sensitive K +-channels K-ATP channels). Luciferase was introduced into HIT M2.2 cells a nd whole pancreatic islets by transient expression of firefly (Photinus pyr alis) luciferase cDNA. In transfected cells, extracellular addition of luci ferin increased the luminescence signal to a maximum within 50-120 s, Addit ion of 1 mu M Of the mitochondrial uncoupler FCCP decreased the luminescenc e, an effect partly reversed upon withdrawal of the compound. High concentr ations of glucose increased cytoplasmic free ATP concentration. Changes in the luminescence signal were accompanied by changes in activity of the ATP- sensitive K+-channel. Transfection per se did not deteroriate cell function , as verified by experiments showing similar changes in cytoplasmic free Ca 2+-concentration, [Ca2+](i), in both transfected and nontransfected cells. By measuring the cytoplasmic ATP concentration and KATP channel activity un der similar experimental conditions, it was possible to establish, for the first time, a direct relationship between these two parameters. This indeed suggests that the cytoplasmic ATP concentration has a crucial role in the regulation of KATP channel activity under physiological conditions, (C) 199 8 Federation of European Biochemical Societies.