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Papers In Press, published online ahead of print November 16, 2001
J. Biol. Chem, 10.1074/jbc.M107984200
Submitted on August 20, 2001
Revised on November 8, 2001
Accepted on November 16, 2001

TGFbeta1 regulates Kir2.3 inward rectifier K+ channels via phospholipase C and protein kinase C-delta in reactive astrocytes from adult rat brain

Pablo R. Perillan, Mingkui Chen, Eric A. Potts, and J. Marc Simard

Department of Neurosurgery, University of Maryland, Baltimore, MD 21201

Corresponding Author: msimard{at}surgery1.umaryland.edu

The multifunctional cytokine, transforming growth factor beta 1 (TGFbeta 1), exerts complex effects on astrocytes with early signaling events being less well characterized than transcriptional mechanisms. We examined the effect of TGFbeta 1 on the 14 pS Kir2.3 inward rectifier K+ channel in rat primary cultured reactive astrocytes. Immunofluorescence study showed that cells co-expressed TGFbeta 1-receptor(R)1 and TGFbeta 1-R2, Kir2.3, and GFAP. Patch clamp study showed that TGFbeta 1 (0.1-100 ng/ml) caused a rapid (<5 min) depolarization due to dose-dependent downregulation of Kir2.3 channels, which was mimicked by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA; 10-500 nM), and which was inhibited by the PKC inhibitor, calphostin C (100 nM), by PKC desensitization produced by 3-hr exposure to PMA (100 nM), and by the PKC-delta isoform-specific inhibitor, rottlerin (50 mM). Immunoblot analysis and confocal imaging showed that TGFbeta 1 caused PKC-delta translocation to membrane, and co-immunoprecipitation experiments showed that TGFbeta 1 enhanced association between Kir2.3 and PKC-delta . Additional electrophysiological experiments showed that Kir2.3 channel downregulation was blocked by the phospholipase C inhibitors, neomycin (100 mM) and D609 (200 mM). Given the commonality of signaling involving PLC-PKC-delta , we speculate that TGFbeta 1–evoked depolarization may be an early signaling event related to gene transcription in astrocytes.


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