SNAP-25, one of the three SNARE-proteins responsible for synaptic release, can be phosphorylated by Protein Kinase C on Ser-187, close to the fusion pore. In neuroendocrine cells, this phosphorylation event potentiates vesicle recruitment into releasable pools, whereas the consequences of phosphorylation for synaptic release remain unclear. We mutated Ser-187 and expressed two mutants (S187C and S187E) in the context of the SNAP-25B-isoform in SNAP-25 knockout glutamatergic autaptic neurons. Whole-cell patch clamp recordings were performed to assess the effect of Ser-187 phosphorylation on synaptic transmission. Blocking phosphorylation by expressing the S187C mutant did not affect synapse density, basic evoked or spontaneous neurotransmission, the readily-releasable pool size or its Ca2+-independent or Ca2+-dependent replenishment. Furthermore, it did not affect the response to phorbol esters, which activate PKC. Expressing S187C in the context of the SNAP-25A isoform also did not affect synaptic transmission. Strikingly, the - potentially phosphomimetic - mutant S187E reduced spontaneous release and release probability, with the largest effect seen in the SNAP-25B isoform, showing that a negative charge in this position is detrimental for neurotransmission, in agreement with electrostatic fusion triggering. During the course of our experiments, we found that higher SNAP-25B expression levels led to decreased paired pulse potentiation, probably due to higher release probabilities. Under these conditions, the potentiation of evoked EPSCs by phorbol esters was followed by a persistent down-regulation, probably due to a ceiling effect. In conclusion, our results indicate that phosphorylation of Ser-187 in SNAP-25 is not involved in modulation of synaptic release by Ca2+ or phorbol esters.

中文翻译：

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Ser187处的SNAP-25磷酸化不参与Ca2 +或佛波酯依赖性突触释放的增强。

SNAP-25是负责突触释放的三种SNARE蛋白之一，可以被蛋白激酶C在靠近融合孔的Ser-187上磷酸化。在神经内分泌细胞中，这种磷酸化事件使囊泡募集到可释放的池中，而磷酸化对突触释放的后果尚不清楚。我们突变了Ser-187，并在SNAP-25基因敲除的谷氨酸能自闭神经元中的SNAP-25B同工型的背景下表达了两个突变体（S187C和S187E）。进行全细胞膜片钳记录以评估Ser-187磷酸化对突触传递的影响。通过表达S187C突变体来阻止磷酸化，不会影响突触密度，基本诱发或自发的神经传递，易释放的库大小或其Ca2 +独立或Ca2 +依赖的补充。此外，它不影响对激活PKC的佛波酯的反应。在SNAP-25A同工型的背景下表达S187C也不会影响突触传递。令人惊讶的是，潜在的拟磷酸化突变体S187E降低了自发释放和释放的可能性，在SNAP-25B亚型中表现出最大的作用，表明该位置的负电荷不利于神经传递，与静电融合触发一致。在我们的实验过程中，我们发现更高的SNAP-25B表达水平导致配对脉冲电位降低，这可能是由于更高的释放概率所致。在这些条件下，佛波酯增强了诱发的EPSC，随后可能由于上限效应而持续下调。综上所述，