Effects Of Interaction Between Rab3A And Synaptotagmin 1 On Neurotransmitter Release In Cultured PC12 Cells

Synaptic transmission is initiated by synaptic vesicle exocytosis,which occurs in several consecutive steps:docking,priming and Ca2+-triggered fusion and release.Although the correct targeting of the synaptic vesicles and the precise spatio-temporal regulation of exocytosis involve the interactions of many proteins,a series of evidences suggest that the critical regulation of this process is mediated by two proteins with opposite actions:synaptotagmin,a Ca2+-sensor that is essential for Ca2+-triggered fusion and release,and Rab3 that limits the number of vesicles that can be fused as a function of Ca2+.Up to now,how these two proteins work oppositely but cooperatively has not been fully understood.Rab3A and synaptotagmin 1 are the most abundant isoforms of Rab3 and synaptotagmin in brain,respectively.In our previous in vitro experiments,the two proteins were for the first time demonstrated to directly interact with each other,thereby participating in the regulation of synaptic membrane fusion and vesicle excytosis.In the present research,we have made a systematic investigation into the interaction of Rab3A and synaptotagmin 1 and its effects on the transmitter release,using digitonin-permeabilised PC 12 cells as model cells and dopamine,the main catecholamine in PC 12 cells as detection index.We first investigated the effects of Rab3A protein on dopamine in PC cells with normal expression of synaptotagmin 1.It was found that there were dopamines secreted from the cells in the presence or absence of Ca2+,suggesting that there were both Ca2+-dependent and Ca2+-independent dopamine release pathways.However,the Ca2+-dependent dopamine release was dominant as the dopamine released in the presence of 10 ?M Ca2+ significantly more than that in the absence of Ca2+(130.3 ng vs 34.5 ng,p<0.01).Furthermore,the effects of Rab3A on the two dopamine release pathways were demonstrated to be different.In the absence of Ca2+,low concentration of Rab3A(500 nM)decreased the release of dopamine whereas higher concentrations(1000 nM-2500 nM)increased the release.However,in the presence of 10 ?M Ca2+,application of different concentrations of Rab3A(500 nM-2500 nM)to the cultured PC 12 cells continuously reduced the amount of secreted dopamine as the concentrations of Rab3A were increased.In order to investigate the involvement of synaptotagmin 1 in the dopamine release and the interaction with Rab3A protein in this process,we effectively inhibited the expression of synaptotagmin 1 using a specific RNAi sequence screened from five candidates,and then comparatively analyzed the effects of Rab3 A on the release of dopamine in the presence and absence of Ca2+.It was found that in the absence of Ca2+ the dopamine releases were not significantly different between before and after synaptotagmin 1 expression inhibition,suggesting that synaptotagmin 1 did not mediated the Ca2+-independent dopamine release.In contrast,Ca2+-dependent dopamine release after RNA interference of synaptotagmin 1 expression was weaker than that before RNA interference(118.1 ng vs 130.3 ng,p<0.01),demonstrating that synaptotagmin 1 was involved in the Ca2+-dependent dopamine release.In the presence of 10 ?M Ca2,treatment of the PC 12 cells with different concentrations of Rab3A(500 nM-2500 nM)led to a gradual decrease in the amount of secreted dopamine.However,the decrease amplitude was obviously reduced(by 18.4%at 2500 nM)compared with that before the expression of synaptotagmin 1 in PC 12 cells was inhibited,demonstrating that the effect of Rab3A on the release of dopamine involved the interaction with synaptotagmin 1.Besides,it was found that the intracellular dopamine content at all above cases remained a relatively stable level,which suggested that there was an effective mechanism maintaining such a stable state in the cells.In addition,for confirming the reliability of the results,we used Cellometer K1 cell function analyzer to analyze the PC12 cells that were treated with digitonin-containing Rab3A protein solution.The results showed that the apoptotic rate and cell cycle in the treated PC 12 cells were not significantly different from those in the control PC 12 cells,demonstrating that the changes in the dopamine release after treatment with Rab3A were not due to the alternations in physiological state of the PC 12 cells.Besides,the transmission electron microscopic observation of the PC 12 cells indicated that the secretion activity of the Rab3A protein-treated PC 12 cells was obviously weakened compared with that of the control PC 12 cells.