Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles.
Classical neurotransmitters such as gamma-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 Ca(2+)-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.
Itakura
M
M
Department of Biological Sciences, Tokyo Institute of Technology, 4259 Nagatuta-cho, Midoriku, Yokohama, 226-8511, Japan.
Misawa
H
H
Sekiguchi
M
M
Takahashi
S
S
Takahashi
M
M
D000109:Acetylcholine / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D005089:Exocytosis / Q000502:physiology*; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000502:physiology*; D016716:PC12 Cells; D011188:Potassium / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013572:Synaptic Vesicles / Q000502:physiology*; D014162:Transfection; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D017931:DNA Primers; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C493459:SLC18A3 protein, human; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; C095992:complexin I; C095993:complexin II; D019382:Human Growth Hormone; D002795:Choline O-Acetyltransferase; D000109:Acetylcholine; D011188:Potassium; D002118:Calcium