Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, VU University, 1081, HV, Amsterdam, The Netherlands.
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Nobels väg 12A, PO Box 281, 171 77, Stockholm, Sweden.
Sci Rep. 2020 Feb 21;10(1):3181. doi: 10.1038/s41598-020-59757-y.
Phosphorylation of Munc18-1 (Stxbp1), a presynaptic organizer of synaptic vesicle fusion, is a powerful mechanism to regulate synaptic strength. Munc18-1 is a proposed substrate for the Down Syndrome-related kinase dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a) and mutations in both genes cause intellectual disability. However, the functional consequences of Dyrk1a-dependent phosphorylation of Munc18-1 for synapse function are unknown. Here, we show that the proposed Munc18-1 phosphorylation site, T479, is among the highly constrained phosphorylation sites in the coding regions of the gene and is also located within a larger constrained coding region. We confirm that Dyrk1a phosphorylates Munc18-1 at T479. Patch-clamp physiology in conditional null mutant hippocampal neurons expressing Cre and either wildtype, or mutants mimicking or preventing phosphorylation, revealed that synaptic transmission is similar among the three groups: frequency/amplitude of mEPSCs, evoked EPSCs, paired pulse plasticity, rundown kinetics upon intense activity and the readily releasable pool. However, synapses expressing the phosphomimic mutant responded to intense activity with more pronounced facilitation. These data indicate that Dyrk1a-dependent Munc18-1 phosphorylation has a minor impact on synaptic transmission, only after intense activity, and that the role of genetic variation in both genes in intellectual disability may be through different mechanisms.
Munc18-1（Stxbp1）的磷酸化是调节突触强度的一种强大机制，它是突触囊泡融合的一种突触前组织者。Munc18-1 是与唐氏综合征相关的双特异性酪氨酸磷酸化调节激酶 1a（Dyrk1a）的潜在底物，这两种基因的突变都会导致智力障碍。然而，Dyrk1a 依赖性 Munc18-1 磷酸化对突触功能的功能后果尚不清楚。在这里，我们表明，拟议的 Munc18-1 磷酸化位点 T479 是基因编码区中高度受约束的磷酸化位点之一，并且也位于更大的受约束编码区内。我们证实 Dyrk1a 在 T479 处磷酸化 Munc18-1。在表达 Cre 的条件性 null 突变海马神经元中的膜片钳生理学，以及表达野生型、模拟或防止磷酸化的突变体的膜片钳生理学，表明三种组之间的突触传递相似：mEPSC 的频率/幅度、诱发的 EPSC、成对脉冲可塑性、强烈活动时的失活动力学和可释放的囊泡库。然而，表达磷酸模拟突变体的突触在强烈活动后表现出更明显的易化。这些数据表明，Dyrk1a 依赖性 Munc18-1 磷酸化对突触传递的影响较小，仅在强烈活动后才会出现，并且这两种基因中的遗传变异在智力障碍中的作用可能通过不同的机制。
