Overexpression of rat neuronal calcium sensor‐1 in rodent NG108‐15 cells enhances synapse formation and transmission

Abstract

The role of rat neuronal calcium sensor‐1 (NCS‐1), a Ca2+‐binding protein, in synapse formation and transmitter release was examined in mouse neuroblastoma × rat glioma hybrid NG108‐15 cells in culture. Wild‐type NG108‐15 cells expressed rodent NCS‐1. Endogenous NCS‐1 was partially co‐localized with the synaptic protein SNAP‐25 at the plasma membrane in both cell bodies and processes, but not with the Golgi marker β‐COP, an individual coat subunit of the coatomer complex present on Golgi‐derived vesicles. In NG108‐15 cells co‐cultured with rat myotubes, partial co‐localization of SNAP‐25 and NCS‐1 was observed at the plasma membrane of neurites and growth cones, some of which had synaptic contacts to muscle cells. Transient co‐transfection of the rat NCS‐1 cDNA and green fluorescent protein (GFP) resulted in NCS‐1 overexpression in about 30 % of the cells as determined by fluorescence microscopy. The rate of functional synapse formation with co‐cultured rat myotubes increased 2‐fold as determined by the presence of miniature endplate potentials (MEPPs) in NCS‐1‐overexpressing NG108‐15 cells compared to non‐ and mock‐transfected cells. The number of neurites per cell, branches per neurite and length of neurites was slightly less in cells that were either transiently transfected (GFP‐NCS‐1‐fluorescence positive) or stably transformed with NCS‐1 compared to GFP‐NCS‐1‐negative, non‐transfected or mock‐transfected NG108‐15 cells. The number of action potentials that elicited endplate potentials increased in NG108‐15 cells stably transformed with rat NCS‐1. The mean number of quanta per impulse (m) increased 5‐fold. These results show that NCS‐1 functions to facilitate synapse formation, probably because of the increased quantal content of evoked acetylcholine release.

Up-regulation of neuronal calcium sensor-1 (NCS-1) in the prefrontal cortex of schizophrenic and bipolar patients

“…It is possible that the over-expression of this protein may affect signal processing in the DLPFC in SCHIZ and BPD patients by promoting the A-type K ϩ current (25), affecting the activity of P͞Q-type Ca 2ϩ channels (26,27) and facilitating synaptic neurotransmitter release (20,22). An increase in the K ϩ current involved in membrane outward rectification would prevent DLPFC neurons from reaching firing threshold (50).…”

“…The regulation of DR desensitization is not the only reported activity of this protein. Among the major actions of NCS-1 and its Drosophilae orthologue, frequenin, are regulation of exocytosis of secretory and neurotransmitter substances (18)(19)(20)(21)(22), control of trafficking of cellular proteins (23,24), and modulation of the activity of K ϩ and Ca 2ϩ ionic channels (25)(26)(27). In addition, up-regulation of the NCS-1 gene has been observed in the rat dentate gyrus after long-term potentiation, suggesting that this protein may be involved in activity-dependent neuronal plasticity (28).…”

Down-regulation of Voltage-gated Ca2+ Channels by Neuronal Calcium Sensor-1 Is β Subunit-specific

“…In these cell types, NCS-1 modulates synaptic transmission (14,22,34), secretion (21,25), or cellular excitability (18,20) via complex processes that include regulation of key enzymes for membrane transport (15,17,23,24,35) and specific regulation of various ion channels (16, 18 -20, 28). K ϩ channels were the first ion channel target to be characterized in expression systems (26,36).…”

Neuronal calcium sensor-1 modulation of optimal calcium level for neurite outgrowth

“…For instance, NCS-1 expression increases in grey matter and decreases in white matter during embryogenesis and early postnatal stages (Kawasaki et al, 2003). Overexpression of NCS-1 in NG108-15/rat myotube co-cultures (Chen et al, 2001), and of the Drosophila homologue Frequenin (Frq) in motor nerve terminals (Angaut-Petit et al, 1998), reduces the number of neurites. More recently, NCS-1 was shown to impede neurite extension without altering branching in NGF-treated PC12 cells by potentiating TRPC5-evoked Ca 2+ currents (Hui et al, 2006).…”