|dc.description.abstracteng||In chemical synapses, neurons communicate with each other via two mechanisms known as fast and slow synaptic transmission. Fast synaptic transmission happens within milliseconds and is triggered by the arrival of an action potential to the nerve terminal, resulting in the release of neurotransmitters. In contrast, the slow synaptic transmission occurs over hundreds of millisecond to minutes and involves elevation of second messengers such as Ca2+ ions and cAMP. Second messengers directly or indirectly modulate activity of kinases and protein phosphatases (PP) and consequently regulate properties of their substrates via phosphorylation/dephosphorylation.
Up to date, the regulation of synaptic transmission by phosphorylation of presynaptic proteins has been studied by targeted and low-throughput approaches. In the current study, we have applied tandem mass spectrometry-based phosphoproteomics workflow to globally investigate the molecular mechanism of slow synaptic transmission through dynamic changes of the nerve terminal protein phosphorylation. Comparison of the stimulated nerve terminals phosphoproteome in the presence and the absence of extracellular Ca2+ ions revealed that Ca2+ plays a major role in the regulation of protein phosphorylation, while membrane depolarization has a minor role. Moreover, it was demonstrated that components of active zone protein group undergo extensive phosphorylation changes during stimulation. Noteworthy many phosphosites of large scaffolding proteins bassoon and piccolo were dephosphorylated.
In the next step, considering the role of Ca2+ in the regulation of phosphosites, the pharmacological specific inhibitors of a Ca2+-dependent kinase (CaMKII) and a phosphatase (Calcineurin) were used to investigate their role in regulation of the phosphosites. Calcineurin inhibition revealed that many of regulated active zone phosphosites are Calcineurin substrates. In addition, it was shown that, phosphorylation of critical phosphosites were downregulated upon CaMKII inhibition.
Overall, our data suggest that upon the stimulation of nerve terminal activity of CaMKII, PKC, PKA, ERK1/2, CK2, PP2A, and Calcineurin is upregulated, whereas activity of CdK5, GSK-3, PP1 is downregulated. In conclusion, we propose a model in which Ca2+ has a primary role in regulation of tightly interconnected network of kinases and phosphatases upon stimulation.||de