Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels.
1. In order to investigate the mechanism underlying MgATP-dependent recovery of ATP-sensitive potassium (KATP) channels, we expressed Kir6.2/SUR2A (inwardly rectifying K+ channel subunit/sulfonylurea receptor) or C-terminal-truncated Kir6.2 (Kir6.2DeltaC26) in COS7 cells (Green monkey kidney cells), and carried out inside-out patch clamp experiments. 2. After patch excision in ATP-free internal solution, the activity of Kir6.2/SUR2A channels could be maximally recovered by the application of 5 mM MgATP. Subsequent application of 100 microM Ca2+ induced a rapid decay of Kir6.2/SUR2A activity to 11.6 +/- 1.1 % (mean +/- s.e.m.) of the control level (Ca2+-induced run-down; n = 64). 3. MgATP (5 mM) recovered 99.4 +/- 4.2 % (n = 13) of the Ca2+-induced run-down. Protein kinase inhibitors such as W-7, H-7, H-8 and genistein did not inhibit this reaction. However, wortmannin, an inhibitor of phosphatidylinositol 3- and 4-kinases, blocked the MgATP-dependent recovery in a concentration-dependent manner; the magnitudes of recovery were 35.7 +/- 7.2 % (10 microM) and 4.3 +/- 2.5 % (100 microM) of the Ca2+-induced run-down. 4. MgUDP (10 mM) reversed the Ca2+-induced run-down of Kir6.2/SUR2A channels by 60.4 +/- 7.6 % (n = 5). Wortmannin failed to modify this reaction. 5. Kir6.2DeltaC26 channels, which opened in the absence of SUR2A, were less sensitive to Ca2+; Kir6.2DeltaC26 channels were inactivated to 44.8 +/- 4.4 % (n = 14) by 100 microM Ca2+. MgATP recovered the Ca2+-induced run-down of Kir6.2DeltaC26 by 89.8 +/- 7. 7 % (n = 9), and 100 microM wortmannin inhibited this reaction (1.8 +/- 2 %, n = 7). 6. Application of 10 microM phosphatidylinositol-4, 5-bisphosphate (PI-4,5-P2) recovered the activity of Kir6.2/SUR2A channels after Ca2+-induced run-down (104.3 +/- 6.4 %, n = 10). Even after the MgATP-dependent recovery was blocked by 100 microM wortmannin, PI-4,5-P2 reactivated the channels (102.3 +/- 8.6 %, n = 5). Similar results were obtained with Kir6.2DeltaC26. 7. These results suggest that the entity of MgATP-dependent recovery may be membrane lipid phosphorylation rather than protein phosphorylation, and that synthesis of PI-4,5-P2 or phosphatidylinositol-3,4, 5-trisphosphate may upregulate Kir6.2 channels.
Xie
L H
LH
Department of Physiology and Biophysics, Graduate School of Medicine, Kyoto University, Japan.
Takano
M
M
Kakei
M
M
Okamura
M
M
Noma
A
A
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000502:physiology*; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004558:Electric Stimulation; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015221:Potassium Channels / Q000187:drug effects*; D024661:Potassium Channels, Inwardly Rectifying; D047428:Protein Kinase Inhibitors; D011955:Receptors, Drug / Q000187:drug effects*; D064233:Sulfonylurea Receptors; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects; D014530:Uridine Diphosphate / Q000494:pharmacology; D000077191:Wortmannin
D018528:ATP-Binding Cassette Transporters; D000730:Androstadienes; D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D047428:Protein Kinase Inhibitors; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D014530:Uridine Diphosphate; D000255:Adenosine Triphosphate; D000077191:Wortmannin