Dynamic Interactions Between G Proteins and Calcium at SNARE Complexes

SNARE 复合物中 G 蛋白和钙之间的动态相互作用

基本信息

批准号：
8578107
负责人：
SIMON T ALFORD
金额：
$ 38.18万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-08 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8578107
关键词：
Acetylcholine Action Potentials Address Aggressive behavior Axon Binding Biological Models Botulinum Toxins Brain Brain Diseases Buffers Calcium Chemicals Communication Complex Data Dependency Depressed mood Disease Dopamine Dyes Exocytosis Exocytosis Inhibition Fluorescence Functional disorder G-Protein-Coupled Receptors GTP-Binding Proteins Glutamates In Situ In Vitro Intervention Ion Channel Label Lampreys Lead Liposomes Location Measurement Measures Mediating Membrane Mental Depression Methods Mind Modeling Nervous system structure Neuraxis Neuromodulator Neuronal Plasticity Neurons Neurosecretion Neurotransmitters Outcome Parkinson Disease Pathology Pathway interactions Physiologic pulse Physiological Preparation Presynaptic Terminals Probability Process Protein Subunits Proteins Role Running S-nitro-N-acetylpenicillamine SNAP receptor Schizophrenia Second Messenger Systems Serotonin Serotonin Receptor 5-HT1B Signal Transduction Site Solutions Sum Synapses Synaptic Transmission Synaptic Vesicles System Vesicle aqueous base flash photolysis fluorophore gamma-Aminobutyric Acid inhibitor/antagonist insight millisecond neuropathology neurotransmission neurotransmitter release postsynaptic presynaptic prevent protein complex protein function protein protein interaction public health relevance receptor receptor function reconstitution relating to nervous system research study second messenger synaptic function synaptotagmin

项目摘要

DESCRIPTION (provided by applicant): Understanding the function of synapses is key to understanding the nervous system, and yet, while synaptic transmission is ubiquitously controlled by presynaptic G protein coupled receptors (GPCRs), the means by which these receptors modify release is contentious. Central nervous diseases, in which GPCR function is treated by pharmacological intervention, cover conditions ranging from aggression, depression and schizophrenia. Inhibitory presynaptic GPCRs alter synaptic function by many mechanisms. These may utilize the G?? G protein subunit to modify presynaptic ion channels and then synaptic secretion, or to modify the very machinery of vesicle fusion, the SNARE complex. Alternatively, these signaling systems may act indirectly through, other second messengers. We have focused on direct mechanisms by which G?? inhibits transmitter release and have demonstrated that G?? can inhibit neurosecretion by an interaction with the fusion machinery - the SNARE complex. This interaction occurs late in the activation of the synapse, after priming, in vesicles whose SNARE complex is formed. We hypothesize that G?? binds to the ternary SNARE complex to compete with Ca2+-dependent synaptotagmin binding. In this way G?? might be thought of as means to interfere with the final switch leading to neurosecretion. We propose that this action of G?? also confers a Ca2+ dependency on G?? -mediated presynaptic inhibition because high presynaptic Ca2+ concentrations allow synaptotagmin to compete more effectively with the machinery of vesicle fusion. The final outcome of this G?? competition with synaptotagmin allows a fine-tuned control of secretion. G?? causes kiss-and-run fusion of the exocytosing vesicle. This reduces the peak synaptic concentration of neurotransmitter, adding a complex twist to the method by which GPCRs alter synaptic function. We propose to investigate competition between the switch that activates synapses - Ca2+-synaptotagmin - and an inhibitor of this process - G??. We hypothesize that G?? and synaptotagmin share an interaction site on the SNARE complex. To demonstrate this, we will investigate G?? competition with synaptotagmin at the SNARE complex and compare effects of Ca2+ on this competition to similar manipulations in a simple reconstituted model for fusion and at the lamprey giant axon in situ. We will then modify in situ SNARE complex proteins using intracellularly applied Botulinum toxins to compare similar truncations in vitro. We will also use fluorescence measurements of exogenously applied G?? model systems of the protein components of vesicle fusion to determine whether Ca2+ evokes similar effects on binding and on neurotransmitter release in situ. These experimental paradigms will then be reproduced with dynamically fluctuating Ca2+ concentrations to mimic a real world activity of central synapses. These experiments will afford insight into the dynamic modulation possible with a simple protein-protein interaction and explain some of the complexities of presynaptic GPCR-mediated presynaptic modulation and their roles in neuropathologies.

描述（由申请人提供）：了解突触的功能是理解神经系统的关键，但是，尽管突触传播被突触前G蛋白偶联受体（GPCR）无处不在，但这些受体修改释放的手段是有争议的。通过药理学干预治疗GPCR功能的中枢神经疾病涵盖了侵略性，抑郁和精神分裂症的疾病。抑制性突触前GPCR通过许多机制改变突触功能。这些可能利用G？ G蛋白亚基修改突触前离子通道，然后突触分泌，或修改囊泡融合的机器，即SNARE复合物。或者，这些信号系统可能会通过其他第二使者间接起作用。我们专注于直接机制？抑制发射机释放，并证明了G？可以通过与融合机械的相互作用 - 圈圈复合物抑制神经分泌。这种相互作用发生在突触后的激活中，在启动后，在形成圈圈复合物的囊泡中。我们假设该G？结合三元snare复合物，与Ca2+依赖性突触抗体结合竞争。这样g ??可能被认为是干扰导致神经分泌的最终开关的手段。我们建议G？还赋予了Ca2+对G的依赖性？ - 介导的突触前抑制作用，因为高突触前Ca2+浓度使突触素蛋白可以与囊泡融合的机械更有效地竞争。这个G的最终结果？与SynaptoTagmin的竞争允许对分泌的微调控制。 g ??导致胞外囊泡的亲吻和运行融合。这降低了神经递质的峰值突触浓度，从而为GPCR改变突触功能的方法增加了复杂的扭曲。我们建议研究激活突触的开关之间的竞争-Ca2+-synaptotagmin-和此过程的抑制剂-g ??。我们假设该G？并在SNARE综合体上共享突触量。为了证明这一点，我们将研究G？在SNARE复合物中与突触量的竞争，并将Ca2+对该竞争的影响与类似的操作进行比较，以融合的简单重组模型和Lamprey Giant Axon Intu。然后，我们将使用细胞内施用的肉毒杆菌毒素来修饰原位圈圈复合蛋白，以比较体外类似的截断。我们还将使用外源应用G的荧光测量值？囊泡融合的蛋白质成分的模型系统，以确定Ca2+是否会引起对结合和对神经递质释放的相似影响。然后，这些实验范式将以动态波动的Ca2+浓度重现，以模仿中央突触的现实世界活动。这些实验将通过简单的蛋白质 - 蛋白质相互作用来洞悉可能的动态调节，并解释突触前GPCR介导的突触前调节的某些复杂性及其在神经病理学中的作用。