Role of tyrosine phosphorylation in leptin activation of ATP-sensitive K+ channels in the rat insulinoma cell line CRI-G1

• Published in: The Journal of physiology 1998-07, Vol.510 (1), p.47-61
• Main Authors: Harvey, J., Ashford, M. L. J.
• Format: Article
• Language: English
• Subjects: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine - pharmacology; Adenosine Triphosphate - pharmacology; Adenylyl Imidodiphosphate - pharmacology; Androstadienes - pharmacology; Animals; Enzyme Inhibitors - pharmacology; Insulinoma - metabolism; Insulinoma - pathology; Leptin; Nitriles - pharmacology; Original; Pancreatic Neoplasms - metabolism; Pancreatic Neoplasms - pathology; Phosphoric Monoester Hydrolases - antagonists & inhibitors; Phosphorylation; Potassium Channels - drug effects; Potassium Channels - physiology; Protein Tyrosine Phosphatases - antagonists & inhibitors; Protein-Tyrosine Kinases - antagonists & inhibitors; Proteins - pharmacology; Rats; Tumor Cells, Cultured; Tyrosine - metabolism; Tyrphostins; Wortmannin
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.1998.047bz.x

Using whole-cell and cell-attached recording configurations, the role of phosphorylation in leptin activation of ATP-sensitive K + (K ATP ) channels was examined in the rat CRI-G1 insulinoma cell line. Whole-cell current clamp recordings demonstrated that, following dialysis with the non-hydrolysable ATP analogue 5′-adenylylimidodiphosphate (AMP-PNP; 3–5 mM), the leptin-induced hyperpolarization and increase in K + conductance were completely inhibited. Under current clamp conditions, application of the broad-spectrum protein kinase inhibitor H-7 (10 μM) had no effect on the resting membrane potential or slope conductance of CRI-G1 insulinoma cells and did not occlude the actions of leptin. Application of the tyrosine kinase inhibitors genistein (10 μM), tyrphostin B42 (10 μM) and herbimycin A (500 nM) all resulted in activation of K ATP channels. In cell-attached recordings, the presence of tyrphostin B42 (10 μM) in the pipette solution activated tolbutamide-sensitive K ATP channels in CRI-G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect. The serine/threonine-specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 μM) did not prevent or reverse leptin activation of K ATP channels. In contrast, whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 μM) prevented the actions of both leptin and tyrphostin B42. In conclusion, leptin activation of K ATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3-kinase (PI 3-kinase) as wortmannin prevented activation of K ATP channels by tyrphostin B42.