title: PIP2 and ATP cooperatively prevent cytosolic Ca2+-induced modification of ATP-sensitive K+ channels in rat pancreatic beta-cells.
abstract: The factors that influence functional coupling between the sulfonylurea receptor (SUR1) and Kir6.2 subunits of ATP-sensitive K+ (K+(ATP)) channels were studied in rat pancreatic beta-cells using patch clamp and microfluorometric techniques. Tolbutamide at 10 micromol/l inhibited K+(ATP) channels in association with occurrence of action currents, but further exposure of beta-cells to the drug for 30 min or longer resulted in reappearance of K+(ATP) channel events. Half-maximal inhibition concentration (IC50) for tolbutamide was 1.5 microl/mol in 2.8 mmol/l glucose, and it was increased to 13.3 micromol/l when the cellular metabolism was inhibited by 0.5 mmol/l 2,4-dinitrophenol (DNP) for 5 min. Tolbutamide at 10 micromol/l induced an increase in cytosolic Ca2+ concentration ([Ca2+]i), and its amplitude was markedly reduced following exposure to 0.5 mmol/l DNP or long-term (30 min) exposure to 10 micromol/l tolbutamide. This tolbutamide insensitivity, as assessed by the [Ca2+]i response, was not observed when the external Ca2+ was omitted during the long-term exposure to tolbutamide. In cell-attached membrane patches, the tolbutamide insensitivity was also produced by treatment of cells with 150 micromol/l diazoxide and 25 mmol/l KCl in the presence, but not absence, of 2 mmol/l Ca2+ in the external solution. When the cytoplasmic face of inside-out membrane patches was treated with higher Ca2+ concentrations (2 micromol/l), both ADP-evoked activation and tolbutamide-induced inhibition of K+ ATP channels were attenuated with retaining ATP-induced inhibition, indicating the modification of K+(ATP) channels. The Ca2+-induced channel modification was prevented partially by phosphatidylinositol 4,5-bisphosphate (PIP2) and completely by ATP and PIP2 together, but not by ATP alone. Treatment of the channel with cytochalasin D, a disrupter of F-actin, evoked channel modification similar to that induced by Ca2+. The modification was prevented completely by phalloidin, a stabilizer of F-actin. In conclusion, long-term exposure to tolbutamide or metabolic inhibition causes modification of K+ ATP channels via mechanisms involving Ca2+-dependent reaction. 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).
authors.affiliation: First Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Koriyama
authors.forename: M
authors.initials: M
authors.lastname: Kakei
authors.forename: M
authors.initials: M
authors.lastname: Nakazaki
authors.forename: K
authors.initials: K
authors.lastname: Yaekura
authors.forename: K
authors.initials: K
authors.lastname: Ichinari
authors.forename: Q
authors.initials: Q
authors.lastname: Gong
authors.forename: S
authors.initials: S
authors.lastname: Morimitsu
authors.forename: T
authors.initials: T
authors.lastname: Yada
authors.forename: C
authors.initials: C
authors.lastname: Tei
mesh_terms: D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000199:Actins / Q000037:antagonists & inhibitors / Q000502:physiology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D015638:Cytochalasin D / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry*; D003981:Diazoxide / Q000494:pharmacology; D004553:Electric Conductivity; D007515:Islets of Langerhans / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010590:Phalloidine / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects* / Q000502:physiology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014044:Tolbutamide / Q000494:pharmacology
chemical_list: D000199:Actins; D019269:Phosphatidylinositol 4,5-Diphosphate; D015221:Potassium Channels; D010590:Phalloidine; D015638:Cytochalasin D; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D014044:Tolbutamide; D003981:Diazoxide; D019297:2,4-Dinitrophenol; D002118:Calcium