Calcineurin involvement in the regulation of high-threshold Ca2+ channels in NG108-15 (rodent neuroblastoma x glioma hybrid) cells.
1. We examined the relationship between calcineurin (protein phosphatase 2B (PP2B) and voltage-operated Ca2+ channels (VOCCs) in NG108-15 cells. PP2B expression in NG108-15 cells was altered by transfection with plasmid constructs containing a full length cDNA of human PP2B beta(3) in sense (CN-15) and antisense (CN-21) orientation. 2. Confocal immunocytochemical localization showed that in wild-type cells, PP2B immunoreactivity is uniformly distributed in undifferentiated cells and located at the inner surface of soma membrane and neurites in differentiated cells. 3. To test the Ca2+ dependence of the VOCC, we used high-frequency stimulation (HFS). The L- and N-type VOCCs decreased by 37 and 52%, respectively, whereas the T-type current was only marginally sensitive to this procedure. FK-506 (2 microM), a specific blocker of PP2B, reduced the inhibition of L- and N-type VOCCs induced by HFS by 30 and 33%, respectively. 4. In CN-15-transfected cells overexpressing PP2B, total high-voltage-activated (HVA) VOCCs were suppressed by about 60% at a test potential of +20 mV. Intracellular addition of EGTA or FK-506 into CN-15-transfected cells induced an up to 5-fold increase of HVA VOCCs. 5. These findings indicate that PP2B activity does not influence the expression of HVA Ca2+ channels, but modulates their function by Ca(2+)-dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylated state under control conditions, are downregulated by PP2B upon stimulation, with the major effect on N-type VOCCs.
Department of Pharmacology, Royal Free Hospital School of Medicine, London, UK. elena@serv.biph.kiev.ua
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Lukyanetz
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Piper
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Sihra
D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism; D019703:Calcineurin / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology*; D004558:Electric Stimulation; D004594:Electrophysiology; D005910:Glioma / Q000378:metabolism; D006801:Humans; D006822:Hybrid Cells; D007150:Immunohistochemistry; D015640:Ion Channel Gating / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018613:Microscopy, Confocal; D009447:Neuroblastoma / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats
D015220:Calcium Channels; D019703:Calcineurin