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

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.

Calcium-dependent inactivation of neuronal calcium channel currents is independent of calcineurin

“…Overexpression of human CaN in rodent neuroblastoma × glioma hybrid cells increased the Ca 2ϩ -dependent inactivation of N-type and, to a lesser degree, L-type Ca 2ϩ channels. 32 A physiological significance of this phenomenon is suggested by the observation that CaN and voltage-gated Ca 2ϩ channels are co-localized in dorsal root ganglion cells. 31 Such a phosphatase-mediated mechanism of inactivation could counteract the well known up-regulation of L-type Ca 2ϩ channels by phosphorylation via cyclic-AMP-dependent protein kinase.…”

Modulation of Calcium-Dependent Inactivation of L-Type Ca2+ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

“…The original model of CDI in Helix aspersa included a dephosphorylation cycle by calcineurin as the fundamental step leading to channel closure [39]. Later, evidence for and against an involvement of calcineurin was found [17], [40], [41], [42], [43], [44], [45]. In TC neurons, application of the calcineurin blocker ascomycin boosts CDI [9], [46].…”

A Constitutive, Transient Receptor Potential-like Ca2+ Influx Pathway in Presynaptic Nerve Endings Independent of Voltage-gated Ca2+ Channels and Na+/Ca2+ Exchange

“…Under these conditions, the cells extend long neurites, each with multiple, large presynaptic-like axonal varicosities, capable of Ca 2ϩ -dependent secretion of acetylcholine (33) and synaptic innervation of rodent myotubes (32). The presynapticlike varicosities contain voltage-gated ion channels typical of presynaptic sites (34,35), synaptic vesicles and mitochondria, and Ca 2ϩ stores (35). Expression of shRNA Constructions in Differentiated NG108-15 Cells-Transfection of mouse shRNA constructs into differentiated NG108-15 cells was accomplished using FuGene (Roche Diagnostics), with which we typically obtain 80 -90% transfection efficiency as gauged by expression of green fluorescent protein.…”