Transforming Growth Factor-β1 Regulates Kir2.3 Inward Rectifier K+ Channels via Phospholipase C and Protein Kinase C-δ in Reactive Astrocytes from Adult Rat Brain

Abstract

The multifunctional cytokine, transforming growth factor ␤ 1 (TGF-␤ 1 ), exerts complex effects on astrocytes with early signaling events being less well characterized than transcriptional mechanisms. We examined the effect of TGF-␤ 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-␤ 1 receptors 1 and 2, Kir2.3, and glial fibrillary acidic protein (GFAP). Patch clamp study showed that TGF-␤ 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-␦ isoform-specific inhibitor rottlerin (50 M). Immunoblot analysis and confocal imaging showed that TGF-␤ 1 caused PKC-␦ translocation to membrane, and co-immunoprecipitation experiments showed that TGF-␤ 1 enhanced association between Kir2.3 and PKC-␦. Additional electrophysiological experiments showed that Kir2.3 channel down-regulation was blocked by the phospholipase C inhibitors, neomycin (100 M) and D609 (200 M). Given

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EGF and HB-EGF modulate inward potassium current in human bladder urothelial cells from normal and interstitial cystitis patients

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