Overexpression of rat neuronal calcium sensor-1 in rodent NG1108-15 cells enhances synapse formation and transmission

1. The role of rat neuronal calcium sensor-1 (NCS-1), a Ca2+-bincling prote in, in synapse formation and transmitter release was examined in mouse neur oblastoma x rat glioma hybrid NG108-15 cells in culture. 2. Wild-type NG108-15 cells expressed rodent NCS-1. Endogenous NCS-1 was pa rtially co-localized with the synaptic protein SNAP-25 at the plasma membra ne in both cell bodies and processes, but not with the Golgi marker beta -C OP, an individual coat subunit of the coatomer complex present on Golgi-der ived vesicles. 3. 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 g rowth cones, some of which had synaptic contacts to muscle cells. 4. Transient co-transfection of the rat NCS-1 cDNA and green fluorescent pr otein (GFP) resulted in NCS-1 overexpression in about 30% of the cells as d etermined by fluorescence microscopy. 5. The rate of functional synapse formation with co-cultured rat myotubes i ncreased 2-fold as determined by the presence of miniature endplate potenti als (MEPPs) in NCS-1-overexpressing NG108-15 cells compared to non- and moc k-transfected cells. 6. The number of neurites per cell, branches per neurite and length of neur ites 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. 7. The number of action potentials that elicited endplate potentials increa sed in NG108-15 cells stably transformed with rat NCS-1. The mean number of quanta per impulse (m) increased 5-fold. 8. These results show that NCS-1 functions to facilitate synapse formation, probably because of the increased quantal content of evoked acetylcholine release.