Real time course of transmitter release of SSVs and LDCVs; SNARE function analysed in synaptobrevin2 and cellubrevin knock out mice

Abstract

Part 1Neurotransmitter release is mediated by Ca2+ dependent exocytosis of synaptic vesicles. The release process has very rapid kinetics. Using carbon fibers as electrochemical detectors, the release of the neurotransmitter serotonin was measured from cultured Retzius cells of the leech (Hirudo medicinalis). The kinetics of amperometric signals reflects the time course of transmitter release and the diffusion of molecules from the release site to the detector. Based on this, an explicit formula, HWDextran = (HWRinger-HWreal) × DRinger/DDextrant + HWreal (here, HW is the half-width of individual amperometric spike, and D is the diffusion coefficient of transmitter molecules) was developped to calculate the real time course (HW) of transmitter release of SSVs (small synaptic vesicles) and LDCVs (large dense core vesicles), by introducing Dextran molecules (40,000 Da) to change the diffusion coefficient. The real time course (HWreal) of transmitter release of SSVs is about 198 ms and the contribution of diffusional broadening on the kinetics of individual amperometric signals of SSVs is about 20 %.These results indicate that exocytotic release of SSVs is non-instantaneous and the majority of the kinetics of individual amperometric signals of SSVs is dominated by real time course of transmitter release. The real time course (HWreal) of the release process of LDCVs was also determined (about 1133 ms). Furthermore, the temperature effects on the release process of SSVs were also investigated. The results show that increasing temperature can accelerate the release kinetics by a Q10 value of 1.77 and leads to a dramatic increase of release frequency by a Q10 value of 6.0. In the end, the underlying mechanism responsible for the temperature effects on the kinetics of amperometric events can be well interpreted by a model developed in this study. It is proposed that, the fusion pore size changes in two steps, the initial opening of the fusion pore which is very rapid, and afterwards the expansion of the pore which is relatively slow. The first step dominates the kinetics of release process, in contrast, the second step of fusion pore dilation contributes less to the kinetics. Compared the first step, the dilation phase is more sensitive to temperature changes, which can explain that the final change of kinetics is only 1.77 fold for temperature change of 10 oC.Part 2SNAREs (soluble NSF-attachment protein receptors) are generally acknowledged as central components of membrane fusion reactions. During fusion, SNAREs (the vesicle SNARE synaptobrevin and the plasma membrane SNAREs syntaxin 1 and SNAP-25) form complexes through their SNARE motifs. Although SNAREs have been studied in great detail, their precise role in membrane fusion has remained unclear. In this study, the exocytosis in the chromaffin cells from synaptobrevin2 (syb2) and cellubrevin (VAMP3, one member of the synaptobrevin / VAMP family of SNAREs) null mice was examined.In the absence of syb2, exocytosis persisted, but the release frequency was decreased by 20 %. Single vesicular fusion events showed normal characteristics of spike phase, but the duration of foot signal was prolonged by two-fold compared to wild type. Expression of TeNT light chain in syb2-/- chromaffin cells completely abolished the residual release. The prolonged foot duration and the decreased release frequency were fully rescued to wild type level by over-expressing syb2 protein. However, over-expression of cellubrevin (VAMP3) in syb2-/- cells had an opposite effect on the foot duration, that exogenous VAMP3 even prolonged the foot duration by 3-fold compared to wild type. The exocytosis in VAMP3-/- chromaffin cells was also examined, no significant differences were found between mutant and control cells, with respect to secretion and individual fusion events. It is concluded that synaptobrevin proteins support exocytosis, and they are functionally redundant. Both synaptobrevin isoforms (syb2 and VAMP3) are involved in the stage of fusion pore dilation, but they have opposite effects on this stage.