REGULATION OF GLUTAMATE STORAGE IN THE SYNAPTIC VESICLE

本研究的目的是扩大对突触囊泡谷氨酸摄取系统的了解，特别是研究其调节。谷氨酸现在被认为是中枢神经系统中主要的兴奋性神经递质。因此，适当的谷氨酸突触传递不仅是基本的神经元交流所必需的，也是学习和记忆形成所必需的。此外，各种证据表明，谷氨酸的异常传递参与了中枢神经系统的多种病理生理过程。囊泡谷氨酸摄取系统是谷氨酸的高度特异性摄取系统，被认为在引导谷氨酸进入神经递质途径而不是代谢途径中起着关键作用。因此，它被假定要受到监管。申请人的实验室先前提供了一种内源性蛋白质因子抑制囊泡谷氨酸摄取的证据。这个因子现在已经被提纯到明显的同质性，这表明它确实是一种有效的蛋白抑制物，显然是突触小泡吸收谷氨酸的特异性抑制物。序列分析表明，这种被称为谷胱甘肽(GSR)的抑制蛋白来自于细胞骨架蛋白的主要成分--α-fodrin。PI建议：(1)通过将不同孵育条件下GSR的产生与[3/H]谷氨酸积聚到突触小泡中的减少相关联，来证明GSR的产生与获得调节囊泡谷氨酸积累的能力之间的关系；(2)通过动力学实验进一步表征GSR的抑制常数；(B)通过检测GSR对囊泡摄取GABA和甘氨酸的影响来确定神经递质的特异性；以及(C)通过检测GSR对H泵ATPase活性、膜电位和谷氨酸流出的影响来确定作用部位；以及(3)证明GSR是在生理条件下，通过不同条件下的突触体孵育，然后通过凝胶印迹放射免疫分析而产生的。这项研究有望有助于更好地了解突触前对谷氨酸传递的调节。人们还希望，这项调查最终将为一些涉及谷氨酸异常传递的神经和精神障碍提供新的见解。

The objective of this research is to broaden understanding of the glutamate uptake system in the synaptic vesicle, particularly to investigate its regulation. Glutamate is now recognized as the major excitatory neurotransmitter in the central nervous system. As such, proper glutamate synaptic transmission is required not only for basic neuronal communication, but also for learning and memory formation. Moreover, a variety of evidence indicates that aberrant glutamate transmission is involved in many types of pathophysiologies in the central nervous system. The vesicular glutamate uptake system is highly specific for glutamate and considered to play a critical role in directing glutamate to the neurotransmitter pathway away from the metabolic pathway. Hence, it has been postulated to be subject to regulation. The applicant's laboratory previously provided evidence for an endogenous proteinaceous factor that inhibits vesicular glutamate uptake. This factor has now been purified to apparent homogeneity, revealing that it is indeed a potent protein inhibitor apparently specific for glutamate uptake into synaptic vesicles. Sequence analyses suggest that this inhibitory protein, referred to as glustoreducin (GSR), is derived from alpha-fodrin, a major cytoskeletal protein component. The PI proposes to (1) demonstrate the relationship between the production of GSR and the acquisition of the ability to regulate vesicular glutamate accumulation, by correlating, under various incubation conditions, the GSR production with the reduction of [3/H]glutamate accumulated into synaptic vesicles; (2) further characterize GSR with respect to (a) the inhibition constant by kinetic experiments; (b) neurotransmitter specificity by examining the effect of GSR on vesicular uptake of GABA and glycine; and (c) site of action by examining the effect on GSR on H+ pump ATPase activity, membrane potential, and glutamate efflux; and (3) demonstrate that GSR is generated from alpha-fodrin under physiologically relevant conditions, by incubating synaptosomes under various conditions, followed by gel blot radioimmunoassay. This research is expected to contribute to a better understanding of presynaptic regulation of glutamate transmission. It is also hoped that this investigation will ultimately provide new insights into some of the neurological and psychiatric disorders involving abnormal glutamate transmission.