Leptin activation of ATP-sensitive K⁺ (KATP) channels in rat CRI-G1 insulinoma cells involves disruption of the actin cytoskeleton

1. The role of the cytoskeleton in leptin-induced activation of ATP-sensitive K⁺ (K_ATP) channels was examined in rat CRI-G1 insulin-secreting cells using patch clamp and fluorescence imaging techniques. 2. In whole cell recordings, dialysis with the actin filament stabiliser phalloidin (10 µM) prevented K_ATP channel activation by leptin. 3. Application of the actin filament destabilising agents deoxyribonuclease type 1 (DNase 1; 50 µg ml^-1) or cytochalasin B (10 µM) to intact cells or inside-out membrane patches also increased K_ATP channel activity in a phalloidin-dependent manner. 4. The anti-microtubule agents nocodazole (10 microM) and colchicine (100 µM) had no effect on K_ATP channel activity. 5. Fluorescence staining of the cells with rhodamine-conjugated phalloidin revealed rapid disassembly of actin filaments by cytochalasin B and leptin, the latter action being prevented by the phosphoinositide 3 (PI 3)-kinase inhibitor LY 294002. 6. Activation of K_ATP channels by the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was also prevented by phalloidin. This is consistent with the notion that leptin activates K_ATP channels in these cells by an increase in PtdIns(3,4,5)P3 or a similar 3-phosphorylated phosphoinositol lipid, resulting in actin filament disruption.