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，特别是对其调控的研究。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需要基本的神经元交流，而且还需要学习和记忆的形成。此外，各种证据表明，谷氨酸异常传递参与中枢神经系统的多种病理生理。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从代谢途径引导到神经递质途径中发挥关键作用。因此，它被认为是受监管的。申请人的实验室先前提供了内源性蛋白因子抑制水疱性谷氨酸摄取的证据。该因子现在已被纯化到明显的同质性，表明它确实是一种有效的蛋白质抑制剂，显然对谷氨酸摄取到突触囊泡具有特异性。序列分析表明，这种被称为葡萄糖储存ducin （GSR）的抑制蛋白来源于α -fodrin，一种主要的细胞骨架蛋白成分。PI提出(1)通过在不同的培养条件下，GSR的产生与突触囊泡中积累的[3/H]谷氨酸的减少之间的相关性，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对(a)抑制常数的影响；(b)通过检测GSR对GABA和甘氨酸囊泡摄取的影响来检测神经递质特异性；(c)通过检查GSR对H+泵atp酶活性、膜电位和谷氨酸外排的影响来确定作用位点；(3)通过在不同条件下培养突触体，然后进行凝胶印迹放射免疫分析，证明GSR是在生理相关条件下由α -fodrin产生的。这项研究有望有助于更好地理解谷氨酸传递的突触前调节。也希望这项研究最终将为一些涉及谷氨酸异常传递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.