Ca 2+ ‐Evoked Transmitter Release Is Inhibited in PC12 Cells That Have Synaptotagmin I Silenced By RNAi

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.