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Calcineurin involvement in the regulation of high‐threshold Ca 2+ channels in NG108–15 (rodent neuroblastoma × glioma hybrid) cells
Author(s) -
Lukyanetz E. A.,
Piper T. P.,
Sihra T. S.
Publication year - 1998
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1998.371bk.x
Subject(s) - transfection , calcineurin , microbiology and biotechnology , chemistry , cell culture , endocrinology , biology , medicine , transplantation , genetics
1 We examined the relationship between calcineurin (protein phosphatase 2B (PP2B)) and voltage‐operated Ca 2+ 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 PP2Bβ 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 Ca 2+ 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. 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 Ca 2+ 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 being on N‐type VOCCs.