MOLECULAR MECHANISMS OF NEUROTRANSMISSION AND ITS MODULATION

神经传递及其调节的分子机制

• 批准号: 6290672
• 负责人: Zu-hang Sheng
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290672
• 关键词: action potentials; calcium channel; calcium flux; guanine nucleotide binding protein; intermolecular interaction; membrane fusion; nerve /myelin protein; neurotransmitter transport; northern blottings; protein isoforms; protein structure function; synaptic vesicles; yeast two hybrid system

The principal research goal of the Synaptic Function Unit is to understand the molecular mechanisms underlying the neurotransmitter release process and its modulation. In past year, we have obtained promising results on the following four projects: (1) Continued characterization of a novel SNARE-associated protein called Snapin, which was isolated in our laboratory in 1998. Snapin is a synaptic vesicle membrane associated protein involved in neurotransmission by directly binding to SNAP-25 and potentiating interactions between the SNARE proteins and synaptotagmin, a key molecular intermediate in Ca2+-dependent exocytosis. Introducing truncated Snapin and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons (SCGNs) in culture reversibly inhibits synaptic transmission. We also identified that several residues on Snapins carboxyl terminal are critical for its binding to SNAP-25. The paper describing our work on cloning and biochemical and physiological characterization of Snapin was published in Nature Neuroscience (2, 119-124, 1999). (2) More recently, we have found that Snapin may be a target for cAMP-dependent protein kinase A (PKA) in the neurotransmitter secretory machinery. We found that both recombinant and native Snapin derived from rat brain synaptosomes are phosphorylated by PKA. Deletion mutation and PCR-based site-directed mutagenetic experiments pinpointed the phosphorylation site to serine50. PKA-phosphorylation of Snapin significantly increases its binding to SNAP-25. A Snapin mutation of serine50 to aspartic acid (S50D) mimics the effect of PKA phosphorylation of Snapin, suggesting that the introduction of a negatively charged residue at serine50 is critical to regulate Snapin?s function. Furthermore, the Snapin S50D mutant enhances the association of synaptotagmin with the SNARE complex. Our results suggest that Snapin is a PKA target for modulating transmitter release and neuronal plasticity via the cAMP-dependent signal transduction pathway. The paper on PKA phosphorylation of Snapin has been submitted. (3) We have cloned and characterized a protein called syntaphilin that interacts with syntaxin-1A. Northern, immunoblotting, and immunocytochemical analyses demonstrated that syntaphilin is selectively expressed in brain and is highly enriched at nerve terminals. In vitro binding and co-immunoprecipitation studies revealed that syntaphilin competes with SNAP-25 for binding to syntaxin-1, and inhibits SNARE complex formation by absorbing free syntaxin-1 at nerve terminals. Transient over-expression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls the amount of free syntaxin-1 available for SNARE complex assembly, and thereby regulates synaptic vesicle exocytosis. Our paper describing the cloning and characterization of syntaphilin is in press at Neuron. (4) Using the yeast two-hybrid system we have isolated another syntaxin-1A binding protein from the human brain cDNA library. The deduced amino acid sequence is identical to a recently cloned gene called SNAP-29. Using the 3-untranslated cDNA fragment as a probe, our Northern blots showed that a major species of mRNA, 4.4 kb in length, was detected in multiple tissues including brain. The specific interaction of SNAP-29 with syntaxin-1A was further confirmed using in vitro binding studies with recombinant proteins and co-immunoprecipitation analysis with co-transfected HEK 293 T cell lysates. The amino terminal half of SNAP-29, which contains a coiled coil domain, is necessary and sufficient for its interaction with syntaxin-1A. Furthermore, SNAP-29 competes with native SNAP-25 for binding to syntaxin-1 and thus inhibits SNARE complex formation at nerve terminals. Introduction of the amino terminal half of SNAP-29 into presynaptic superior cervical ganglion neurons (SCGNs) in culture significantly inhibits synaptic transmission. Although SNAP-29 has been suggested to be a general SNARE component in membrane trafficking, our findings suggest that it may function as a cytoplasmic regulator for the assembly of the SNARE complex and participate in the docking/fusion process of synaptic vesicle exocytosis. The manuscript describing our work on SNAP-29 is in preparation.

突触功能单元的主要研究目标是了解神经递质释放过程及其调节的分子机制。在过去的一年中，我们在以下四个项目中获得了令人鼓舞的结果：（1）持续表征一种称为Snapin的新型小军斗相关蛋白，该蛋白在1998年在我们的实验室中分离出来。Snapin是一种突触囊泡膜相关的蛋白Ca2+依赖性胞吐作用。在培养物中引入了含有SNAP-25结合序列的截短的Snapin和肽中的SNAP-25结合序列（SCGN）中的突触前宫颈上神经节神经元（SCGN）可逆地抑制突触传播。我们还确定，Snapins羧基末端的几种残基对于与SNAP-25的结合至关重要。该论文描述了我们关于Snapin的克隆和生化和生理特征的工作，在自然神经科学中发表（2，119-124，1999）。 （2）最近，我们发现Snapin可能是神经递质分泌机械中cAMP依赖性蛋白激酶A（PKA）的靶标。我们发现，源自大鼠脑突触体的重组和天然Snapin均被PKA磷酸化。删除突变和基于PCR的位置定向诱变实验将磷酸化位点列为丝氨酸50。 Snapin的PKA磷酸化显着增加了其与SNAP-25的结合。丝氨酸对天冬氨酸（S50D）的Snapin突变模拟了Snapin的PKA磷酸化的作用，这表明在丝网膜上引入负电荷的残基对于调节Snapin的功能至关重要。此外，Snapin S50D突变体增强了突触毒素与军鼓复合物的缔合。我们的结果表明，Snapin是通过CAMP依赖性信号转导途径调节发射机释放和神经元可塑性的PKA靶标。已经提交了有关Snapin的PKA磷酸化的论文。 （3）我们已经克隆并表征了一种称为Syntaphilin的蛋白质，该蛋白质与义话1a相互作用。北部，免疫印迹和免疫细胞化学分析表明，征素蛋白在大脑中有选择地表达，并且在神经末端高度富集。体外结合和共免疫沉淀研究表明，Syntaphilin与SNAP-25竞争以与Syntaxin-1结合，并通过在神经末端吸收游离的自由语法-1来抑制SNARE复合物的形成。培养的海马神经元中征素蛋白的瞬时过表达可显着降低神经递质的释放。此外，在培养物中将征素蛋白引入突触前宫颈神经元中会抑制突触传播。这些发现表明，征素蛋白可以充当分子夹，该分子夹控制可用于圈套复合物组装的游离语法1的量，从而调节突触囊泡外胞胞菌病。我们的论文描述了含义素的克隆和表征，在神经元的印刷中。 （4）使用酵母双杂交系统，我们从人脑cDNA文库中分离了另一种语法1A结合蛋白。推导的氨基酸序列与最近克隆的基因相同，称为SNAP-29。我们的北面印迹使用3个非翻译cDNA片段作为探针，表明在包括大脑在内的多个组织中检测到了一个主要的mRNA，长度为4.4 kb。使用重组蛋白的体外结合研究进一步证实了SNAP-29与Snap-29与语法素-1a的特异性相互作用，并与共转染的HEK 293 T细胞裂解物进行了共免疫沉淀分析。 SNAP-29的氨基端子一半，其中包含盘绕的线圈结构域，对于与Syntaxin-1a的相互作用是必要且足够的。此外，SNAP-29与天然SNAP-25竞争与Syntaxin-1结合，从而抑制神经末端的SNARE复合物的形成。将SNAP-29的氨基末端引入培养物中突触前宫颈神经神经元（SCGN）中的氨基末端会显着抑制突触传播。尽管SNAP-29被认为是膜运输中的一般圈圈成分，但我们的发现表明，它可能是胞质调节剂，用于组装SNARE复合物，并参与突触/融合过程的突触囊泡外胞菌病的融合过程。描述我们在SNAP-29上工作的手稿正在准备。