Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
Cell Res. 2011 May;21(5):741-53. doi: 10.1038/cr.2011.29. Epub 2011 Feb 15.
Stimulus-induced exocytosis of large dense-core vesicles (LDCVs) leads to discharge of neuropeptides and fusion of LDCV membranes with the plasma membrane. However, the contribution of LDCVs to the properties of the neuronal membrane remains largely unclear. The present study found that LDCVs were associated with multiple receptors, channels and signaling molecules, suggesting that neuronal sensitivity is modulated by an LDCV-mediated mechanism. Liquid chromatography-mass spectrometry combined with immunoblotting of subcellular fractions identified 298 proteins in LDCV membranes purified from the dorsal spinal cord, including G-protein-coupled receptors, G-proteins and other signaling molecules, ion channels and trafficking-related proteins. Morphological assays showed that δ-opioid receptor 1 (DOR1), β2 adrenergic receptor (AR), G(αi2), voltage-gated calcium channel α2δ1 subunit and P2X purinoceptor 2 were localized in substance P (SP)-positive LDCVs in small-diameter dorsal root ganglion neurons, whereas β1 AR, Wnt receptor frizzled 8 and dishevelled 1 were present in SP-negative LDCVs. Furthermore, DOR1/G(αi2)/G(β1γ5)/phospholipase C β2 complexes were associated with LDCVs. Blockade of the DOR1/G(αi2) interaction largely abolished the LDCV localization of G(αi2) and impaired stimulation-induced surface expression of G(αi2). Thus, LDCVs serve as carriers of receptors, ion channels and preassembled receptor signaling complexes, enabling a rapid, activity-dependent modulation of neuronal sensitivity.
刺激诱导的大致密核心囊泡 (LDCVs) 的胞吐作用导致神经肽的释放和 LDCV 膜与质膜融合。然而，LDCVs 对神经元膜性质的贡献在很大程度上仍不清楚。本研究发现，LDCVs 与多种受体、通道和信号分子相关，表明神经元敏感性受 LDCV 介导的机制调节。液相色谱-质谱联用与亚细胞级分的免疫印迹鉴定了从背根神经节纯化的 LDCV 膜中的 298 种蛋白质，包括 G 蛋白偶联受体、G 蛋白和其他信号分子、离子通道和运输相关蛋白。形态学测定表明，δ-阿片受体 1 (DOR1)、β2 肾上腺素能受体 (AR)、G(αi2)、电压门控钙通道α2δ1 亚基和 P2X 嘌呤能受体 2 位于小直径背根神经节神经元中 SP 阳性 LDCVs 中，而 β1 AR、Wnt 受体卷曲 8 和盘绕蛋白 1 存在于 SP 阴性 LDCVs 中。此外，DOR1/G(αi2)/G(β1γ5)/磷脂酶 C β2 复合物与 LDCVs 相关。DOR1/G(αi2)相互作用的阻断在很大程度上消除了 G(αi2)的 LDCV 定位，并损害了刺激诱导的 G(αi2)表面表达。因此，LDCVs 作为受体、离子通道和预组装受体信号复合物的载体，能够快速、依赖于活性地调节神经元敏感性。
