Overexpression of rat neuronal calcium sensor-1 in rodent NG108-15 cells enhances synapse formation and transmission.
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 x 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 [beta]-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.
Chen
X L
XL
Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan.
Zhong
Z G
ZG
Yokoyama
S
S
Bark
C
C
Meister
B
B
Berggren
P O
PO
Roder
J
J
Higashida
H
H
Jeromin
A
A
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D002450:Cell Communication / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects; D018920:Coculture Techniques; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D005910:Glioma; D049452:Green Fluorescent Proteins; D020439:Growth Cones / Q000737:chemistry / Q000502:physiology; D006822:Hybrid Cells; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D009045:Motor Endplate / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D016501:Neurites / Q000737:chemistry / Q000502:physiology; D009447:Neuroblastoma; D051580:Neuronal Calcium-Sensor Proteins; D009474:Neurons / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D009479:Neuropeptides / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D013569:Synapses / Q000737:chemistry / Q000502:physiology*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D014162:Transfection
D002135:Calcium-Binding Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; C101464:frequenin calcium sensor proteins; D049452:Green Fluorescent Proteins; D003994:Bucladesine; D000109:Acetylcholine