Modulation of Kv1.5 currents by Src tyrosine phosphorylation: potential role in the differentiation of astrocytes.
Using biophysical techniques, we previously have implicated outwardly rectifying potassium currents in the proliferation of cultured spinal cord astrocytes and have demonstrated that delayed rectifier potassium currents (I(Kd)), in particular, are upregulated on entry into the cell cycle and downregulated with cell cycle exit and differentiation. In the present study, using specific antibodies and antisense oligodeoxynucleotides, we show that this proliferation-dependent potassium current is mediated by the Shaker potassium channel Kv1.5. Downregulation of Kv1.5 protein by antisense oligodeoxynucleotides reduces astrocyte proliferation by approximately 50%, although no observed changes occur in Kv1.5 protein expression during spontaneous differentiation in culture. Tyrosine phosphorylation of Kv1.5, however, is downregulated markedly in differentiated cells but unaltered on cell cycle arrest. Using immunoprecipitation, we show that Kv1.5 is associated with Src family protein tyrosine kinases and that this association does not change with cell differentiation. Inhibition of kinase activity with the Src-specific inhibitor PP2 decreases Kv1.5 phosphorylation, reduces I(Kd), and inhibits astrocyte proliferation, specifically in the G(0)/G(1) phase of cell cycle. Conversely, I(Kd) are potentiated when active Src is present in the pipette. Transfection of quiescent astrocytes with constitutively active Src (Src Y529F) causes marked upregulation of astrocyte proliferation. These data suggest that Kv1. 5 is phosphorylated constitutively by Src kinases during growth and that downregulation of Src activity may underlie both astrocyte differentiation and the accompanying changes in delayed rectifier potassium channel activity.
MacFarlane
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Department of Neurobiology, University of Alabama, Birmingham, Birmingham, Alabama 35294-0021, USA.
Sontheimer
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D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D051668:Delayed Rectifier Potassium Channels; D015536:Down-Regulation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017136:Ion Transport / Q000502:physiology; D051666:Kv1.5 Potassium Channel; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011233:Precipitin Tests; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D051668:Delayed Rectifier Potassium Channels; D004791:Enzyme Inhibitors; C496983:Kcna5 protein, rat; D051666:Kv1.5 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011743:Pyrimidines; D014443:Tyrosine; D019061:src-Family Kinases; D011188:Potassium