Center for Synaptic Neuroscience and Technology, Istituto Italiano Di Tecnologia, Largo Rosanna Benzi 10, 16132, Genoa, Italy.
Department of Experimental Medicine, University of Genova, Viale Benedetto XV, 3, 16132, Genoa, Italy.
Cell Mol Life Sci. 2022 Nov 21;79(12):600. doi: 10.1007/s00018-022-04631-5.
Synapsin I (SynI) is a synaptic vesicle (SV)-associated phosphoprotein that modulates neurotransmission by controlling SV trafficking. The SynI C-domain contains a highly conserved ATP binding site mediating SynI oligomerization and SV clustering and an adjacent main Ca binding site, whose physiological role is unexplored. Molecular dynamics simulations revealed that the E373K point mutation irreversibly deletes Ca binding to SynI, still allowing ATP binding, but inducing a destabilization of the SynI oligomerization interface. Here, we analyzed the effects of this mutation on neurotransmitter release and short-term plasticity in excitatory and inhibitory synapses from primary hippocampal neurons. Patch-clamp recordings showed an increase in the frequency of miniature excitatory postsynaptic currents (EPSCs) that was totally occluded by exogenous Ca chelators and associated with a constitutive increase in resting terminal Ca concentrations. Evoked EPSC amplitude was also reduced, due to a decreased readily releasable pool (RRP) size. Moreover, in both excitatory and inhibitory synapses, we observed a marked impaired recovery from synaptic depression, associated with impaired RRP refilling and depletion of the recycling pool of SVs. Our study identifies SynI as a novel Ca buffer in excitatory terminals. Blocking Ca binding to SynI results in higher constitutive Ca levels that increase the probability of spontaneous release and disperse SVs. This causes a decreased size of the RRP and an impaired recovery from depression due to the failure of SV reclustering after sustained high-frequency stimulation. The results indicate a physiological role of Ca binding to SynI in the regulation of SV clustering and trafficking in nerve terminals.
突触结合蛋白 I（Synapsin I，SynI）是一种突触囊泡（SV）相关磷酸蛋白，通过控制 SV 转运来调节神经递质传递。SynI 的 C 结构域包含一个高度保守的 ATP 结合位点，介导 SynI 寡聚化和 SV 聚集，以及一个相邻的主要 Ca 结合位点，但其生理作用尚未被探索。分子动力学模拟表明，E373K 点突变不可逆地删除了 Ca 与 SynI 的结合，仍然允许 ATP 结合，但诱导 SynI 寡聚化界面的不稳定性。在这里，我们分析了这种突变对原代海马神经元兴奋性和抑制性突触中神经递质释放和短期可塑性的影响。膜片钳记录显示，微小兴奋性突触后电流（miniature excitatory postsynaptic currents，mEPSCs）的频率增加，这完全被外源性 Ca 螯合剂阻断，并伴有静息末端 Ca 浓度的持续增加。诱发的 EPSC 幅度也降低，这是由于易释放池（readily releasable pool，RRP）大小减小所致。此外，在兴奋性和抑制性突触中，我们观察到从突触抑制中恢复明显受损，这与 RRP 再填充受损和 SV 再循环池耗竭有关。我们的研究将 SynI 确定为兴奋性末梢中的一种新型 Ca 缓冲剂。阻止 Ca 与 SynI 的结合会导致更高的基础 Ca 水平，从而增加自发释放的概率并分散 SV。这会导致 RRP 减小，并由于持续高频刺激后 SV 再聚类失败，导致从抑制中恢复受损。结果表明，Ca 与 SynI 的结合在调节神经末梢 SV 聚类和转运中具有生理作用。
