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Originally published In Press as doi:10.1074/jbc.M107984200 on November 16, 2001

J. Biol. Chem., Vol. 277, Issue 3, 1974-1980, January 18, 2002
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Transforming Growth Factor-beta 1 Regulates Kir2.3 Inward Rectifier K+ Channels via Phospholipase C and Protein Kinase C-delta in Reactive Astrocytes from Adult Rat Brain*

Pablo R. PerillanDagger §, Mingkui ChenDagger §, Eric A. PottsDagger , and J. Marc SimardDagger §||

From the Departments of Dagger  Neurosurgery, § Pathology, and  Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

The multifunctional cytokine, transforming growth factor beta 1 (TGF-beta 1), exerts complex effects on astrocytes with early signaling events being less well characterized than transcriptional mechanisms. We examined the effect of TGF-beta 1 on the 14-pS Kir2.3 inward rectifier K+ channel in rat primary cultured reactive astrocytes. Immunofluorescence study showed that cells co-expressed TGF-beta 1 receptors 1 and 2, Kir2.3, and glial fibrillary acidic protein (GFAP). Patch clamp study showed that TGF-beta 1 (0.1-100 ng/ml) caused a rapid (<5 min) depolarization because of dose-dependent down-regulation of Kir2.3 channels, which was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (10-500 nM) and which was inhibited by the PKC inhibitor calphostin C (100 nM), by PKC desensitization produced by 3 h of exposure to phorbol 12-myristate 13-acetate (100 nM), and by the PKC-delta isoform-specific inhibitor rottlerin (50 µM). Immunoblot analysis and confocal imaging showed that TGF-beta 1 caused PKC-delta translocation to membrane, and co-immunoprecipitation experiments showed that TGF-beta 1 enhanced association between Kir2.3 and PKC-delta . Additional electrophysiological experiments showed that Kir2.3 channel down-regulation was blocked by the phospholipase C inhibitors, neomycin (100 µM) and D609 (200 µM). Given the commonality of signaling involving PLC-PKC-delta , we speculate that TGF-beta 1-evoked depolarization may be an early signaling event related to gene transcription in astrocytes.

* This work was supported by a Merit Review Grant from the Department of Veterans Affairs (Baltimore, MD).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Neurosurgery, 22 South Greene St., Baltimore, MD 21201. Tel.: 410-328-0850; Fax: 410-328-0756; E-mail: msimard@surgery1.umaryland.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.