TY - JOUR
T1 - Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles
AU - Itakura, Makoto
AU - Misawa, Hidemi
AU - Sekiguchi, Mariko
AU - Takahashi, Seiichi
AU - Takahashi, Masami
N1 - Funding Information:
This work was supported in part by grants from the Human Frontier Science Program (RG-79/96), by Grant-in-Aid 07279105 for Scientific Research in Priority Areas on “Functional Development of Neuronal Circuits”, the Ministry of Education, Science Sports and Culture of Japan to M.T., and by a fellowship from the Japan Society for the Promotion of Science for Young Scientists to M.I. We thank Dr. M. Seagar for critical reading of the manuscript and Ms. S. Yamamori for technical assistance.
PY - 1999/11/30
Y1 - 1999/11/30
N2 - Classical neurotransmitters such as γ-aminobutyric acid and glutamate are released from synaptic nerve terminals by exocytosis of synaptic vesicles. PC12 cells also have SSVs capable of storing acetylcholine (ACh). A novel method to examine the effect of transient transfection of any gene of interest on the exocytosis of SSVs was developed. The transfection of choline acetyltransferase (ChAT) into PC12 cells which have lost ACh synthesizing activity resulted in the accumulation of a substantial amount of ACh. Synthesized ACh was released in Ca2+-dependent manner. Release was thought to occur by an exocytosis of SSVs because: (1) release was abolished by treating the cells with vesamicol, a specific inhibitor of the vesicular ACh transporter (VAChT) localizing specifically in SSVs; and (2) the release was further increased by cotransfecting rat VAChT with the ChAT. By means of this method, we showed that overexpression of complexin I or II with ChAT markedly suppressed high-K+-dependent ACh release of SSVs.
AB - Classical neurotransmitters such as γ-aminobutyric acid and glutamate are released from synaptic nerve terminals by exocytosis of synaptic vesicles. PC12 cells also have SSVs capable of storing acetylcholine (ACh). A novel method to examine the effect of transient transfection of any gene of interest on the exocytosis of SSVs was developed. The transfection of choline acetyltransferase (ChAT) into PC12 cells which have lost ACh synthesizing activity resulted in the accumulation of a substantial amount of ACh. Synthesized ACh was released in Ca2+-dependent manner. Release was thought to occur by an exocytosis of SSVs because: (1) release was abolished by treating the cells with vesamicol, a specific inhibitor of the vesicular ACh transporter (VAChT) localizing specifically in SSVs; and (2) the release was further increased by cotransfecting rat VAChT with the ChAT. By means of this method, we showed that overexpression of complexin I or II with ChAT markedly suppressed high-K+-dependent ACh release of SSVs.
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U2 - 10.1006/bbrc.1999.1756
DO - 10.1006/bbrc.1999.1756
M3 - Article
C2 - 10600482
AN - SCOPUS:0033619769
SN - 0006-291X
VL - 265
SP - 691
EP - 696
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -