REGULATION OF GLUTAMATE STORAGE IN THE SYNAPTIC VESICLE

本研究的目的是拓宽对突触囊泡中谷氨酸摄取系统的理解，特别是研究其调控。谷氨酸现在被认为是中枢神经系统中的主要兴奋性神经递质。因此，适当的谷氨酸突触传递不仅是基本神经元通信所必需的，而且也是学习和记忆形成所必需的。此外，各种证据表明，异常谷氨酸传输参与中枢神经系统中的许多类型的病理生理学。囊泡谷氨酸摄取系统对谷氨酸具有高度特异性，并被认为在将谷氨酸引导至远离代谢途径的神经递质途径中起关键作用。因此，它被假定为受到管制。申请人的实验室先前提供了抑制囊泡谷氨酸摄取的内源性蛋白质因子的证据。这个因子现在已经被纯化到明显的同质性，这表明它确实是一种有效的蛋白质抑制剂，对突触囊泡摄取谷氨酸具有明显的特异性。序列分析表明，这种抑制蛋白，称为glustoreducin（GSR），是来自α-胞衬蛋白，一个主要的细胞骨架蛋白组分。PI建议（1）通过在各种孵育条件下将GSR产生与突触囊泡中蓄积的[3/H]谷氨酸减少相关联，证明GSR产生与获得调节囊泡谷氨酸蓄积能力之间的关系;（2）进一步表征GSR（a）通过动力学实验确定抑制常数;（B）通过检查GSR对GABA和甘氨酸的囊泡摄取的作用的神经递质特异性;和（c）通过检查GSR对H+泵ATP酶活性、膜电位和谷氨酸流出的作用的作用位点;和（3）通过在各种条件下孵育突触体，然后进行凝胶印迹放射免疫测定。这一研究有助于更好地理解谷氨酸传递的突触前调控。也希望这项调查最终将提供新的见解，一些神经和精神疾病涉及异常谷氨酸传输。

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.