Institute of Clinical Chemistry, University Medical Center Goettingen, Goettingen, Germany; Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.
Mol Cell Proteomics. 2021;20:100061. doi: 10.1016/j.mcpro.2021.100061. Epub 2021 Feb 12.
Synaptic transmission is mediated by the regulated exocytosis of synaptic vesicles. When the presynaptic membrane is depolarized by an incoming action potential, voltage-gated calcium channels open, resulting in the influx of calcium ions that triggers the fusion of synaptic vesicles (SVs) with the plasma membrane. SVs are recycled by endocytosis. Phosphorylation of synaptic proteins plays a major role in these processes, and several studies have shown that the synaptic phosphoproteome changes rapidly in response to depolarization. However, it is unclear which of these changes are directly linked to SV cycling and which might regulate other presynaptic functions that are also controlled by calcium-dependent kinases and phosphatases. To address this question, we analyzed changes in the phosphoproteome using rat synaptosomes in which exocytosis was blocked with botulinum neurotoxins (BoNTs) while depolarization-induced calcium influx remained unchanged. BoNT-treatment significantly alters the response of the synaptic phoshoproteome to depolarization and results in reduced phosphorylation levels when compared with stimulation of synaptosomes by depolarization with KCl alone. We dissect the primary Ca-dependent phosphorylation from SV-cycling-dependent phosphorylation and confirm an effect of such SV-cycling-dependent phosphorylation events on syntaxin-1a-T21/T23, synaptobrevin-S75, and cannabinoid receptor-1-S314/T322 on exo- and endocytosis in cultured hippocampal neurons.
突触传递是通过突触小泡的受调控胞吐作用来介导的。当传入动作电位使突触前膜去极化时，电压门控钙离子通道打开，导致钙离子内流，触发突触小泡（SVs）与质膜融合。SVs 通过内吞作用进行回收。突触蛋白的磷酸化在这些过程中起着主要作用，有几项研究表明，突触磷酸蛋白质组在去极化时迅速变化。然而，尚不清楚这些变化中哪些与 SV 循环直接相关，哪些可能调节其他也受钙依赖性激酶和磷酸酶控制的突触前功能。为了解决这个问题，我们使用大鼠突触体分析了磷酸蛋白质组的变化，在突触体中，使用肉毒杆菌神经毒素（BoNTs）阻断胞吐作用，而去极化诱导的钙离子内流保持不变。BoNT 处理显著改变了突触磷酸蛋白质组对去极化的反应，与单独用 KCl 去极化刺激突触体相比，磷酸化水平降低。我们从 SV 循环依赖性磷酸化中分离出主要的 Ca 依赖性磷酸化，并在培养的海马神经元中证实了这种 SV 循环依赖性磷酸化事件对突触融合蛋白 1a-T21/T23、突触融合蛋白 S75 和大麻素受体 1-S314/T322 在外排和内吞作用中的作用。
