CALCINEURIN INVOLVEMENT IN THE REGULATION OF HIGH-THRESHOLD CA2-15 (RODENT NEUROBLASTOMA X GLIOMA HYBRID) CELLS( CHANNELS IN NG108)

1. We examined the relationship between calcineurin (protein phosphata se 2B (PP2B)) and voltage-operated Ca2+ channels (VOCCs) in NG108-15 c ells. PP2B expression in NG108-15 cells was altered by transfection wi th plasmid constructs containing a full length cDNA of human PP2B beta (3) in sense (CN-15) and antisense (CN-21) orientation. 2. Confocal im munocytochemical localization showed that in wild-type cells, PP2B imm unoreactivity is uniformly distributed in undifferentiated cells and l ocated at the inner surface of soma membrane and neurites in different iated 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 a nd 52%, respectively, whereas the T-type current was only marginally s ensitive to this procedure. FK-506 (2 mu M), a specific blocker of PP2 B, reduced the inhibition of L- and N-type VOCCs induced by HFS by 30 and 33%, respectively. 4. In CN-15-transfected cells overxpressing PP2 B, total high-voltage-activated (HVA) VOCCs were suppressed by about 6 0% 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 infl uence the expression of HVA C2+ channels, but modulates their function by C2+-dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylat ed state under control conditions, are downregulated by PP2B upon stim ulation, with the major effect being on N-type VOCCs.