G protein-mediated modulation of synaptic vesicle fusion

描述(由申请人提供)：突触传递效率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。众所周知，位于突触前终末的受体可以深刻地抑制神经递质的释放。其中，G蛋白偶联受体是无处不在的。我们已经证明，G蛋白偶联受体通过直接在囊泡融合的核心复合体上介导的效应来抑制突触传递。GBG直接与SNAP-25结合，SNAP-25并入启动囊泡的SNARE复合体中。我们现在假设，这种调制的机制是随着突触小泡向突触间隙释放递质而改变融合孔动力学。这种传输方式的改变可能会对突触传输的实际工作方式和我们对其工作的解释产生深远的影响。瞬时融合，将在重复活动中保存小泡，也可能通过有利于不同的突触后受体具有不同的亲和力和不同的突触下位置来改变突触传递的方式。在这个方案中，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。我们将使用融合突触小泡的苯乙酰染料标记来量化突触前G蛋白引起的突触前末端不完全小泡融合事件的持续时间。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内神经递质的时序和浓度的影响。我们还将确定改变这些参数将对不同突触后趋性离子受体的相对激活产生的影响。最后，我们将利用高速成像技术研究突触前G蛋白对个体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.