G protein-mediated modulation of synaptic vesicle fusion

描述（由申请人提供）：突触传递效能的调节是神经可塑性的关键组成部分，而神经可塑性又是大脑的一个决定性特征。位于突触前末端的受体被认为能严重抑制神经递质释放。其中，G蛋白偶联受体无处不在。我们已经证明G蛋白偶联受体通过直接介导囊泡融合核心复合物的作用抑制突触传递。Gbg直接结合入SNARE复合物的SNAP-25。我们现在假设这种调节的机制是突触囊泡释放递质进入突触间隙时融合孔动力学的改变。这种传递方式的变化可能会对突触传递的实际工作方式和我们对其工作方式的理解产生深远的影响。瞬时融合在重复活动中保存囊泡，也可能改变突触传递模式，有利于具有不同亲和力和不同亚突触位置的不同突触后受体。在这个建议中，我们试图确定突触前G蛋白偶联受体调节神经递质释放的机制。我们将量化由突触前G蛋白引起的突触前末端不完全囊泡融合事件的持续时间，使用苯乙烯染料标记融合的突触囊泡。我们还希望通过测量和模拟突触后受体激活的动力学来确定突触前受体激活对突触间隙中神经递质时间和浓度的影响。我们还将确定改变这些参数对不同突触后嗜离子受体的相对激活的影响。最后，我们将利用高速成像技术研究突触前G蛋白对单个突触接触特性的影响。

DESCRIPTION (provided by applicant): Modulation of the efficacy of synaptic transmission is a key component of neural plasticity, which is, in turn, a defining feature of the brain. Receptors located on the presynaptic terminal are known to profoundly depress neurotransmitter release. Of these, G protein-coupled receptors are ubiquitous. We have demonstrated that G protein-coupled receptors inhibit synaptic transmission by an effect mediated directly at the core complex for vesicle fusion. Gbg binds directly to SNAP-25 incorporated into the SNARE complex of primed vesicles. We now hypothesize that the mechanism for this modulation is an alteration in fusion pore dynamics as the synaptic vesicle releases transmitter into the synaptic cleft. This change in mode of transmission is likely to have profound consequences both to the way synaptic transmission actually works, and to the way we interpret it as working. Transient fusion, will conserve vesicles during repetitive activity, it may also change the mode of synaptic transmission by favoring different postsynaptic receptors with different affinities and different subsynaptic locations. In this proposal we seek to determine the mechanism by which presynaptic G protein-coupled receptors modulate release of neurotransmitter. We will quantify the duration of incomplete vesicle fusion events from the presynaptic terminal caused by presynaptic G proteins using styryl dye labeling of fusing synaptic vesicles? We also wish to determine the affects of presynaptic receptor activation on the timecourse and concentration of neurotransmitter in the synaptic cleft by measuring and modeling the kinetics of postsynaptic receptor activation. We will also determine the effects that altering these parameters will impose on the relative activation of different postsynaptic ionotropic receptors. Finally, we will investigate the affect of presynaptic G proteins on the properties of individual synaptic contacts using high-speed imaging techniques.