Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, VU University, 1081 HV, Amsterdam, The Netherlands.
Sylics (Synaptologics BV), PO box 71033, 1008 BA, Amsterdam, The Netherlands.
Sci Rep. 2017 Sep 7;7(1):10817. doi: 10.1038/s41598-017-10936-4.
CAPS (calcium-dependent activator protein for secretion) are multi-domain proteins involved in regulated exocytosis of synaptic vesicles (SVs) and dense core vesicles (DCVs). Here, we assessed the contribution of different CAPS-1 domains to its subcellular localization and DCV exocytosis by expressing CAPS-1 mutations in four functional domains in CAPS-1/-2 null mutant (CAPS DKO) mouse hippocampal neurons, which are severely impaired in DCV exocytosis. CAPS DKO neurons showed normal development and no defects in DCV biogenesis and their subcellular distribution. Truncation of the CAPS-1 C-terminus (CAPS Δ654-1355) impaired CAPS-1 synaptic enrichment. Mutations in the C2 (K428E or G476E) or pleckstrin homology (PH; R558D/K560E/K561E) domain did not. However, all mutants rescued DCV exocytosis in CAPS DKO neurons to only 20% of wild type CAPS-1 exocytosis capacity. To assess the relative importance of CAPS for both secretory pathways, we compared effect sizes of CAPS-1/-2 deficiency on SV and DCV exocytosis. Using the same (intense) stimulation, DCV exocytosis was impaired relatively strong (96% inhibition) compared to SV exocytosis (39%). Together, these data show that the CAPS-1 C-terminus regulates synaptic enrichment of CAPS-1. All CAPS-1 functional domains are required, and the C2 and PH domain together are not sufficient, for DCV exocytosis in mammalian CNS neurons.
钙依赖性分泌激活蛋白（CAPS）是一种多功能蛋白，参与突触小泡（SVs）和致密核心囊泡（DCVs）的受调控分泌。在此，我们通过在 CAPS-1/-2 缺失突变（CAPS DKO）小鼠海马神经元的四个功能域中表达 CAPS-1 突变，评估了不同 CAPS-1 结构域对其亚细胞定位和 DCV 胞吐作用的贡献，CAPS DKO 神经元在 DCV 胞吐作用中严重受损。CAPS DKO 神经元表现出正常的发育，DCV 发生和它们的亚细胞分布没有缺陷。CAPS-1 C 端截断（CAPS Δ654-1355）损害了 CAPS-1 的突触富集。C2 结构域（K428E 或 G476E）或pleckstrin 同源（PH；R558D/K560E/K561E）结构域的突变则没有。然而，所有突变体都挽救了 CAPS DKO 神经元中的 DCV 胞吐作用，仅恢复至野生型 CAPS-1 胞吐作用能力的 20%。为了评估 CAPS 对两种分泌途径的相对重要性，我们比较了 CAPS-1/-2 缺失对 SV 和 DCV 胞吐作用的影响大小。使用相同（强烈）的刺激，与 SV 胞吐作用（39%）相比，DCV 胞吐作用受到相对强烈的损害（96%抑制）。总的来说，这些数据表明 CAPS-1 的 C 端调节 CAPS-1 的突触富集。在哺乳动物中枢神经系统神经元中，CAPS-1 的所有功能域都需要，并且 C2 和 PH 结构域一起是不足以进行 DCV 胞吐作用的。
