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Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP‐dependent recovery of Kir6.2/SUR2A channels
Author(s) -
Xie LaiHua,
Takano Makoto,
Kakei Masafumi,
Okamura Midori,
Noma Akinori
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.655ad.x
Subject(s) - wortmannin , kir6.2 , chemistry , biophysics , sulfonylurea receptor , patch clamp , phosphatidylinositol , kinase , protein subunit , biochemistry , receptor , biology , gene
1 In order to investigate the mechanism underlying MgATP‐dependent recovery of ATP‐sensitive potassium (K ATP ) channels, we expressed Kir6.2/SUR2A (inwardly rectifying K + channel subunit/sulfonylurea receptor) or C‐terminal‐truncated Kir6.2 (Kir6.2ΔC26) 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 μM Ca 2+ induced a rapid decay of Kir6.2/SUR2A activity to 11·6 ± 1·1 % (mean ± s.e.m.) of the control level (Ca 2+ ‐induced run‐down; n = 64). 3 MgATP (5 mM) recovered 99·4 ± 4·2 % ( n = 13 ) of the Ca 2+ ‐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 μM) and 4·3 ± 2·5 % (100 μM) of the Ca 2+ ‐induced run‐down. 4 MgUDP (10 mM) reversed the Ca 2+ ‐induced run‐down of Kir6.2/SUR2A channels by 60·4 ± 7·6 % ( n = 5 ). Wortmannin failed to modify this reaction. 5 Kir6.2ΔC26 channels, which opened in the absence of SUR2A, were less sensitive to Ca 2+ ; Kir6.2ΔC26 channels were inactivated to 44·8 ± 4·4 % ( n = 14 ) by 100 μM Ca 2+ . MgATP recovered the Ca 2+ ‐induced run‐down of Kir6.2ΔC26 by 89·8 ± 7·7 % ( n = 9 ), and 100 μM wortmannin inhibited this reaction (1·8 ± 2 %, n = 7). 6 Application of 10 μM phosphatidylinositol‐4,5‐bisphosphate (PI‐4,5‐P 2 ) recovered the activity of Kir6.2/SUR2A channels after Ca 2+ ‐induced run‐down (104·3 ± 6·4 %, n = 10). Even after the MgATP‐dependent recovery was blocked by 100 μM wortmannin, PI‐4,5‐P 2 reactivated the channels (102·3 ± 8·6 %, n = 5). Similar results were obtained with Kir6.2ΔC26. 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‐P 2 or phosphatidylinositol‐3,4,5‐trisphosphate may upregulate Kir6·2 channels.