REGULATION OF GLUTAMATE STORAGE IN THE SYNAPTIC VESICLE

本研究的目的是扩大对突触小泡中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通讯所必需的，而且也是学习和记忆形成所必需的。此外，各种证据表明异常的谷氨酸传输与中枢神经系统的多种病理生理学有关。囊泡谷氨酸摄取系统对谷氨酸具有高度特异性，并被认为在引导谷氨酸远离代谢途径进入神经递质途径方面发挥着关键作用。因此，它被认为受到监管。申请人的实验室先前提供了抑制囊泡谷氨酸摄取的内源蛋白质因子的证据。该因子现已被纯化至明显的同质性，表明它确实是一种有效的蛋白质抑制剂，明显对突触小泡的谷氨酸摄取具有特异性。序列分析表明，这种抑制蛋白被称为 glustoreducin (GSR)，源自α-胞嘧啶，一种主要的细胞骨架蛋白成分。 PI建议（1）通过在不同的孵化条件下将GSR的产生与突触小泡中积累的[3/H]谷氨酸的减少相关联，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关于(a)抑制常数的特征； (b) 通过检查 GSR 对囊泡摄取 GABA 和甘氨酸的影响来确定神经递质特异性； (c) 通过检查 GSR 对 H 泵 ATP 酶活性、膜电位和谷氨酸流出的影响来确定作用位点； (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，证明GSR是在生理相关条件下由α-胞质蛋白产生的。这项研究预计将有助于更好地理解谷氨酸传输的突触前调节。人们还希望这项研究最终能为一些涉及谷氨酸传输异常的神经和精神疾病提供新的见解。

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.