Leptin activation of ATP-sensitive K+ (KATP) channels in rat CRI-G1 insulinoma cells involves disruption of the actin cytoskeleton
- 1The role of the cytoskeleton in leptin-induced activation of ATP-sensitive K+ (KATP) channels was examined in rat CRI-G1 insulin-secreting cells using patch clamp and fluorescence imaging techniques.
- 2In whole cell recordings, dialysis with the actin filament stabiliser phalloidin (10 μm) prevented KATP channel activation by leptin.
- 3Application 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 KATP channel activity in a phalloidin-dependent manner.
- 4The anti-microtubule agents nocodazole (10 μm) and colchicine (100 μm) had no effect on KATP channel activity.
- 5Fluorescence 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.
- 6Activation of KATP 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 KATP 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.