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Ca 2+ ‐Evoked Transmitter Release Is Inhibited in PC12 Cells That Have Synaptotagmin I Silenced By RNAi
Author(s) -
Roden William H,
Papke Jason B.,
Cahill Anne L.,
Macarthur Heather,
Harkins Amy B.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1210-b
Subject(s) - synaptotagmin i , synaptotagmin 1 , vesicular monoamine transporter 2 , synaptic vesicle , microbiology and biotechnology , chemistry , gene knockdown , dopamine , transfection , vesicle , biology , biochemistry , endocrinology , dopamine transporter , dopaminergic , apoptosis , membrane , gene
Many proteins are postulated to function in vesicle release during synaptic transmission. Synaptotagmin I (syt I) is believed to be a primary Ca 2+ sensor protein for Ca 2+ ‐dependent release of vesicles during synaptic transmission. To elucidate how a protein functions as a Ca 2+ sensor, we have specifically and stably eliminated syt I from a model secretory PC12 cell line using a plasmid‐based RNAi system to silence expression of syt I. Immunocytochemistry confirms the specificity and extent of syt I protein knockdown compared to control untransfected cells and cells stably transfected with the plasmid that lacks an insert. In response to 50 mM K + stimulation, stimulated fractional release of both dopamine and norepinephrine was significantly reduced by ~50% in syt I‐shRNA cells when measured by HPLC‐electrochemical detection. In addition, stimulated release of ATP was reduced ~50% as measured by HPLC‐fluorometric detection. Immunocytochemistry showed that neither tyrosine hydroxylase, the rate‐limiting enzyme in the dopamine synthesis pathway, nor vesicular monoamine transporter 2 (VMAT2) which transports dopamine into vesicles, had altered protein expression levels. In contrast to both catecholamine and ATP release, neuropeptide Y stimulated release was abolished in syt I knockdown cells. The differing extent of inhibition of neurotransmitter secretion indicates that syt I may be differentially localized within vesicle populations in PC12 cells.