PIP2 and ATP cooperatively prevent cytosolic Ca2+-induced modification of ATP-sensitive K+ channels in rat pancreatic beta-cells

The factors that influence functional coupling between the sulfonylurea rec eptor (SUR1) and Kir6.2 subunits of ATP-sensitive K+ (K-ATP(+)) channels we re studied in rat pancreatic beta -cells using patch clamp and microfluorom etric techniques. Tolbutamide at 10 mu mol/l inhibited K-ATP(+), channels i n association with occurrence of action currents, but further exposure of b eta -cells to the drug for 30 min or longer resulted in reappearance of K-A TP(+) channel events. Half-maximal inhibition concentration (IC50) for tolb utamide was 1.5 mu mol/l in 2.8 mmol/l glucose, and it was increased to 13. 3 mu mol/l when the cellular metabolism was inhibited by 0.5 mu mol/l 2,4-d initrophenol (DNP) for 5 min. Tolbutamide at 10 mu mol/l induced an increas e in cytosolic Ca2+ concentration ([Ca2+](i)), and its amplitude was marked ly reduced following exposure to 0.5 mmol/l DNP or long-term (30 min) expos ure to 10 mu mol/l tolbutamide. This tolbutamide insensitivity, as assessed by the [Ca2+](i) response, was not observed when the external Ca2+ was omi tted during the long-term exposure to tolbutamide. In cell-attached membran e patches, the tolbutamide insensitivity was also produced by treatment of cells with 150 mu mol/l diazoxide and 25 mmol/l KCI in the presence, but no t absence, of 2 mmol/l Ca2+ In the external solution. When the cytoplasmic face of inside-out membrane patches was treated with higher Ca2+ concentrat ions (2 mu mol/l), both ADP-evoked activation and tolbutamide-induced inhib ition of K-ATP(+) channels were attenuated with retaining ATP-induced inhib ition, indicating the modification of K-ATP(+) channels. The Ca2+-induced c hannel modification was prevented partially by phosphatidylinositol 4,5-bis phosphate (PIP2) and completely by ATP and PIP2 together, but not by ATP al one. Treatment of the channel with cytochalasin D, a disrupter of F-actin, evoked channel modification similar to that induced by Ca2+. The modificati on was prevented completely by phalloidin, a stabilizer of F-actin. In conc lusion, long-term exposure to tolbutamide or metabolic inhibition causes mo dification of K-ATP(+) channels via mechanisms involving Ca2+-dependent rea ction. The modification, which may reflect functional disconnection between SUR1 and Kir6.2, is prevented by ATP and PIP2, which may act cooperatively to stabilize membrane cytoskeletons (F-actin structures).