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

J. Harvey and M. L. J. Ashford

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

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