Dynamic Interactions Between G Proteins and Calcium at SNARE Complexes

描述（由申请人提供）：了解突触的功能是了解神经系统的关键，然而，虽然突触传递普遍由突触前G蛋白偶联受体（gpcr）控制，但这些受体调节释放的方式存在争议。中枢神经疾病中，GPCR功能可通过药物干预治疗，包括攻击性、抑郁症和精神分裂症等病症。抑制性突触前gpcr通过多种机制改变突触功能。这些可能会用到G?？G蛋白亚基修饰突触前离子通道，进而修饰突触分泌，或修饰囊泡融合机制，SNARE复合体。或者，这些信号系统可能通过其他第二信使间接起作用。我们关注的是G?？抑制递质释放，并证明G?？可以通过与融合机制- SNARE复合物的相互作用来抑制神经分泌。这种相互作用发生在突触激活的后期，在启动后，在形成SNARE复合物的囊泡中。我们假设G?？与三元SNARE复合物结合以与Ca2+依赖性突触结合蛋白竞争。以这种方式？？可能被认为是干扰导致神经分泌的最终开关的手段。我们认为G?？也赋予Ca2+依赖于G?？-介导的突触前抑制，因为高突触前Ca2+浓度允许synaptotagmin更有效地与囊泡融合机制竞争。G的最终结果是什么？与synaptotagmin的竞争允许对分泌进行微调控制。G ? ?引起囊泡的亲跑融合。这降低了神经递质的突触峰值浓度，给gpcr改变突触功能的方法增加了一个复杂的转折。我们建议研究激活突触的开关- Ca2+-synaptotagmin -和这一过程的抑制剂- G之间的竞争。我们假设G?？和synaptotagmin在SNARE复合体上共享一个相互作用位点。为了证明这一点，我们将研究G?？在SNARE复合体与synaptotagmin竞争，并比较Ca2+对这种竞争的影响，在一个简单的融合重建模型和在原位的七鳃鳗巨大轴突上进行类似的操作。然后，我们将使用细胞内应用肉毒杆菌毒素来原位修饰SNARE复合物蛋白，以比较体外类似的截断。我们还将使用外源施加G??的荧光测量。囊泡融合蛋白组分的模型系统，以确定Ca2+是否对结合和神经递质原位释放产生类似的影响。这些实验范例将在动态波动的Ca2+浓度下重现，以模拟真实世界的中枢突触活动。这些实验将深入了解简单的蛋白质-蛋白质相互作用的动态调节，并解释突触前gpcr介导的突触前调节的一些复杂性及其在神经病理学中的作用。

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.