Calcineurin involvement in the regulation of high‐threshold Ca2+ channels in NG108–15 (rodent neuroblastoma × glioma hybrid) cells

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Abstract
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 PP2Bβ3 in sense (CN‐15) and antisense (CN‐21) orientation. 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. 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 μM), a specific blocker of PP2B, reduced the inhibition of L‐ and N‐type VOCCs induced by HFS by 30 and 33 %, respectively. 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. These findings indicate that PP2B activity does not influence the expression of HVA Ca2+ channels, but modulates their function by Ca2+‐dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylated state under control conditions, are downregulated by PP2B upon stimulation, with the major effect being on N‐type VOCCs.