CHARACTERIZATION OF THE DROSOPHILA SYNAPTOTAGMIN FAMILY

果蝇突触结合蛋白家族的特征

• 批准号: 6394483
• 负责人: J. TROY LITTLETON
• 金额: $ 28.96万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6394483
• 关键词: Drosophilidae; calcium channel; electrophysiology; epilepsy; exocytosis; gene expression; genetic manipulation; green fluorescent proteins; laboratory rabbit; laboratory rat; membrane transport proteins; mutant; neural plasticity; neural transmission; neurotransmitter transport; phospholipids; polymerase chain reaction; protein isoforms; protein structure function; single strand conformation polymorphism; site directed mutagenesis; synaptotagmin; tissue /cell culture; transmission electron microscopy

The goal of this project is to understand the mechanisms by which the synaptotagmin family functions in neuronal exocytosis, membrane trafficking and synaptic plasticity. Synaptic transmission has evolved as a highly specialized form of cellular vesicle trafficking capable of rapid calcium triggered exocytotic cycles. Modulation of neurotransmitter release in the presynaptic nerve terminal can alter the functional properties of neuronal circuits, leading to dramatic changes in behavioral outputs. Alterations in synaptic strength are also likely to underlie a variety of forms of synaptic plasticity associated with learning and memory and may have important roles in pathological states such as epilepsy, Alzheimer's disease, Parkinson's disease, psychoses and the trinucleotide repeat disorders. Synaptotagmin I is a calcium-binding synaptic vesicle protein that has been shown to play an essential, yet undefined, role in synapse function, possibly serving as the calcium sensor for synaptic exocytosis. Synaptotagmin IV is a unique member of the synaptotagmin family and an intermediate early gene that is upregulated in response to seizures. We have identified a family of two neuronal specific synaptotagmins (syt I and syt IV) and two ubiquitous synaptotagmins (syt V and syt VII) in Drosophila that form an evolutionarily conserved synaptotagmin family from invertebrates to humans. Syt IV is co-expressed on synaptic vesicles with syt I, but contains an evolutionarily conserved amino acid change in the first calcium-binding domain that abolishes its ability to bind membranes in a calcium-dependent manner. Thus, synaptic vesicles contain calcium-dependent and -independent synaptotagmin isoforms, indicating that modulation of the expression of these two isoforms or their function could provide a, unique mechanism for controlling synaptic output during episodes of synaptic plasticity or in response to seizure activity. Our analysis will combine genetic manipulations available in Drosophila with molecular, biochemical, electrophysiological and morphological approaches to investigate this possibility and define the function of the synaptotagmin family in neurotransmission. We will also perform structure-function studies using synaptotagmin I and IV mutants to determine the precise role of each isoform. The results of these studies should significantly advance our understanding of the mechanisms of synapse function and plasticity and provide novel insights into potential pathways that modulate neuronal communication.

该项目的目的是了解突触毒素家族在神经元胞外增生，膜运输和突触可塑性中的机制。 突触传播已演变为一种高度专业的细胞囊泡运输形式，能够快速钙触发胞外循环。神经递质在突触前神经末端释放的调节可以改变神经元电路的功能特性，从而导致行为输出的巨大变化。突触强度的改变也可能是与学习和记忆有关的各种形式的突触可塑性的基础，并且可能在病理状态中具有重要作用，例如癫痫，阿尔茨海默氏病，帕金森氏病，精神病和三核苷酸重复疾病。 Synaptotagmin I是一种钙结合突触囊泡蛋白，已被证明在突触功能中起着必不可少但不确定的作用，可能是突触外胞菌病的钙传感器。 Synaptotagmin IV是突触毒素家族的独特成员，也是一个中间的早期基因，该基因响应于癫痫发作。 我们已经在果蝇中确定了两个神经元特异性突触毒素（SYT I和SYT IV）和两个普遍存在的突触毒素（SYT V和SYT VII）的家族，该家族形成了一种进化保守的突触毒素家族。 SYT IV在突触囊泡上与SYT I共表达，但在第一个钙结合结构域中包含进化保守的氨基酸变化，以废除其以钙依赖性方式结合膜的能力。 因此，突触囊泡含有钙依赖性和非依赖性突触型同工型，表明这两种同工型的表达的调节或它们的功能可以提供一种独特的机制，以控制突触可变性或响应癫痫发作的突触可变性期间突触输出。 我们的分析将将果蝇中可用的遗传操作与分子，生化，电生理和形态学方法相结合，以研究这种可能性，并定义了神经传递中突触毒素家族的功能。我们还将使用SynaptoTagmin I和IV突变体进行结构功能研究，以确定每种同工型的精确作用。 这些研究的结果应大大提高我们对突触功能和可塑性机制的理解，并为调节神经元通信的潜在途径提供新的见解。