Discussion

It has been show that endogenous NCS-1 expresses in the bovine chromaffin cells by previous studies (McFerran et al., 1998) including immunostaining in our lab (Wang, 2010; Chen. 2012). Therefore, I examine the influence of NCS-1 by overexpressed NCS-1 and its mutant forms in bovine chromaffin cell and compared with the GFP group in exocytosis and Ca²⁺ response. The untransfected cells are normal chromaffin cells; I used the group as a comparison for transfection effect. The results show that the cells with transfection have more releases; one possible reason is that the LTX reagents altered the vesicle pool. However, consider the functions of proteins, there are significant differences among the GFP group, NCS-1 group and other mutant groups. That is worthwhile to debate the functions of NCS-1.

Consequently, I focusd the discussions on these cells overexpressing constructs in the following sections.

4.1 Cells overexpressing NCS-1 maintains the exocytosis activities

Cells overexpressing NCS-1 have no significant change when comparing with GFP in amplitude, area, spike numbers and total area. That indicates the contents of neurotransmitters per vesicle, the exocytosis events per cell and the total release contents per cell are similar to the normal level; the uptaking of transmitters and the

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vesicle recruitment are taken as usual. NCS-1 makes moderate functions in the process of vesicle recycling. In addition, there is also no significant change between NCS-1 and GFP in the features of vesicle fusion kinetics: rise time, decay time and half width. That means the size and shape of a spike in NCS-1are matching with GFP.

At the results of spike numbers or total area per stimulation, the contribution to exocytosis in different sweeps in NCS-1 is identical to GFP. It presents that the vesicle pool supplied sufficient vesicles to release as primary; the vesicle recycling works stably. In summary, NCS-1 maintains the exocytosis activities.

4.2 Cells overexpressing NCS-1

or NCS-1

down-regulate the exocytosis

The results of amplitude, area, spike numbers and total area show that NCS-1^G2A or NCS-1^R102Q reduces exocytosis. The contents of neurotransmitters per vesicle, the exocytosis events per cell and the total release contents per cell are smaller to the normal level; the uptaking of transmitters and the vesicle recruitment may be attenuated. The mutant proteins have deficient structures and weaken the primary function of NCS-1 in vesicle recycling. However, in the results of rise time, decay time and half width, there is no significant change when comparing with GFP. These mutants do not change the fusion kinetics of vesicle. At the results of spike numbers or total area per stimulation, the contribution to exocytosis in the 3^rd sweep in

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NCS-1^G2A or NCS-1^R102Q is smaller to GFP. Because the evoked exocytosis is vigorous at the beginning, repetitive stimulations let cell not recover completely.

Therefore the difference in exocytosis events occurs in later stimulations. It presents that NCS-1^G2A and NCS-1^R102Q affect the vesicle recycling during the repetitive stimulations.

The NCS-1^G2A mutant has no plasma membrane anchoring capability and it distributes over the cytosol. It indicates that the capability of membrane targeting is important for the regulation of the normal exocytosis activities. The NCS-1^R102Q mutant has a structural deficit but it still can bind to Ca²⁺ and target the membrane; it may affect the interaction with some proteins to reduce exocytosis.

4.3 Cells overexpressing NCS-1

have an elevated exocytosis level

Cells overexpressing NCS-1^E120Q have significantly large area when comparing with GFP. It represents that NCS-1^E120Q may increases the uptaking of transmitters.

The results of the fusion kinetics indicate cells overexpressing NCS-1^E120Q have the significantly long decay time which expresses the long duration of vesicle fusion after the maximum release. Besides, the results of spike numbers or total area per stimulation, the contribution to exocytosis in different sweeps in NCS-1^E120Q is identical to GFP. It presents that NCS-1^E120Q keeps the primary vesicle pool; the

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vesicle recycling works stably. These results suggest that even without the ability of Ca²⁺ binding, NCS-1^E120Q may target some proteins to raise the contents and prolong the fusion time of the vesicle.

4.4 The timing of exocytosis events

Through the accumulation spikes figure (Fig. 7), it can be known the precise occurring timing of spikes. Except the NCS-1^G2A group, the accumulation spikes curves are exponential distribution in other groups. In general, the evoked exocytosis is vigorous after stimulation and then becomes weak gradually over time. But the curve of NCS-1^G2A group is near linear, it has a relative uniform distribution of spikes.

The possible explanation is that NCS-1^G2A down- regulates the exocytosis therefore spike numbers are low in all the timings and there are no obvious accumulation in the beginning.

4.5 NCS-1

overexpression reduces Ca

response

The calcium imaging by Fura-2 reflect the [Ca²⁺]_i change. The results show only the NCS-1^G2A group has a significantly small calcium response. This is possible that NCS-1^G2A affecting the vesicle recycling and reducing the contents of the synaptic vesicle through the reduction of Ca²⁺ response. In contrast, NCS-1 and other mutant

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forms have similar Ca²⁺ response when comparing with the GFP group. Therefore the Ca²⁺ response may be related to the membrane binding capability. Ca²⁺ sources include extracellular Ca²⁺ influx and internal Ca²⁺ stored in ER; NCS-1 and the mutant forms may not only influence the vesicle release, but also mediate the ER release.

4.6 NCS-1 keeps the machinery between exocytosis and Ca

response.

It has been known that Ca²⁺ induces the exocytosis, and NCS-1 also takes part in exocytosis. Therefore I combined the results of my two experiments to draw a relation figure and consider the effect of NCS-1and other mutants. Fig. 10 A and B plots the changes in the Ca²⁺ response against the total spike number and the summarized area.

The mutant forms change the primary machinery between exocytosis and Ca²⁺

response. As it shows that the arrangement of the 6 points is not linear. The relationship between exocytosis and Ca²⁺ response may be indirect through the regulation of the other mutants. These results show that there are a main group includes GFP, NCS-1and NCS-1^E120Q; it means there are similar machinery between exocytosis and Ca²⁺ response in these treatments. It is could be infer that the machinery is similar in the main group. However, it is need to be support by more clear evidences.

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4.7 Internal Ca

store may mediate the function of NCS-1

To clarify the role of NCS-1 in vesicle recycling, I summarized my results to previous studies of capacitance recording and Ca²⁺ current in our lab (Wang, 2010;

Chen, 2012). It shows that there are no significant changes in Ca²⁺ current and [Ca²⁺]i

in NCS-1, and it maintains the exocytosis level, but the membrane capacitance (Cm) is smaller. It could be that NCS-1 promotes more endocytosis event of vesicle. The results of NCS-1^G2A show that it down-regulate the Ca²⁺ current, [Ca²⁺]i, Cm and the exocytosis activities. The results of NCS-1^E120Q show that it down-regulates the Ca²⁺

current and Cm, but it maintains [Ca²⁺]i and have elevated exocytosis level. The Ca²⁺

sources include the influx of extracellular Ca²⁺ and the release of internal Ca²⁺ store.

It may be that the ER store increases the Ca²⁺ release in cytosol to conserve [Ca²⁺]_i; NCS-1^E120Q raises the endocytosis level leads to the decrease of C_m. Last, the results of NCS-1^R102Q show that it down-regulates the Ca²⁺ current, C_m and the exocytosis activities and maintains [Ca²⁺]_i. It may be caused by the increase of Ca²⁺ store from ER.

In conclusion, it is possible that NCS-1 is also related to the internal Ca²⁺ store according to the studies of our lab. Internal Ca²⁺ store may mediate the function of NCS-1. The examination of future works in Ca²⁺ store could be taken by the inhibition of Ca²⁺ channel on the ER membrane.

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4.8 Suggestions for improvements

The results of amperometry recording are different from different methods of analysis. It has been reported that when comparing amperometric spike characteristics between groups of cells, it is more appropriate to compare samples of mean spike values (Colliver et al., 2000). So I presented the mean of spike characteristics per spike per cell as results. I found that there were certain variances in the spike numbers every cell in the same group; the variances were due to the conditions of detection.

The detection of exocytosis events are not only related to the release of vesicle but also the recording of carbon fiber electrode. The skill of manipulation would affect the recoding. Hence the increase of sample numbers could have more data with adequate spike numbers per cell and eliminate the inaccuracy of data. The future researchers might process the data of amperometry severely.

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