Characterization of polyamine transport systems and modulation of NMDA receptor by polyamines

1.通过测量突触囊泡、突触体和神经胶质细胞中的多胺运输以及海马切片中精胺的释放来检测多胺（精胺和亚精胺）在大脑中的循环。突触体和胶质细胞中存在膜电位依赖的多胺转运系统，突触囊泡中存在质子梯度依赖的多胺转运系统。发现精胺在突触囊泡中积累，并通过去极化从大鼠海马切片中释放出来。多胺可能在大脑中起神经调节剂的作用。细胞外质子抑制NMDA受体。为了确定质子敏感性的分子决定因素，我们使用了NR1亚基的扫描诱变。大多数影响质子敏感性的突变集中在第二跨膜结构域的胞外端和邻近连接NR1甘氨酸结合结构域S2部分的连接体上。质子传感器和受体门可以在结构上更统一，功能上更集成。PotA是由PotA、B、C和D组成的亚精胺优先摄取系统的一个组成部分，使用纯化的PotA和PotABC复合物研究了PotA的atp酶活性。当PotA与PotB和PotC结合时，PotA对亚精胺的抑制更加敏感，提示亚精胺具有反馈抑制剂的作用。nh_2末端结构域包含ATP结合袋和ATP酶活性中心。PotA的con -末端结构域包含一个调节atp酶活性的位点和一个参与亚精胺抑制atp酶活性的位点。研究了尸体碱转运蛋白CadB在小马埃希菌体内的功能：CadB同时具有尸体碱摄取和尸体碱排泄活性。在酸性pH下，含有cadB和cadA编码赖氨酸脱羧酶的操纵子在赖氨酸存在下被诱导，导致质子动力的产生。cadBA操纵子在酸性环境下的细胞调控中起重要作用

1.Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. Membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depeolarization. Polyamines may function as neuromodulators in the brain.2.Extracellular protons inhibit NMDA receptors. To identify the molecular determinants of proton sensitivity, we used scanning mutagenesis of NR1 subunit. The majority of mutations that affected proton sensitivity were clustered in the extracellular end of the second transmembrane domain and adjacent linker leading to the S2 portion of the glycine binding domain of NR1. The proton sensor and receptor gate may be structu … More rally and functionally integrated.3.The ATPase activity of PotA, a component of the spermidine-preferential uptake system consisting of PotA, B, C, and D, was studied using purified PotA and a PotABC complex. When PotA was associated with PotB and PotC, PotA became more sensitive to inhibition by spermidine, suggesting that spermidine functions as a feedback inhibitor. The NH_2-terminal domain contains the ATP binding pocket andt he active center of ATPase. The COON-terminal domain of PotA contains a site that regulates ATPase activity and a site involved in the spermidine inhibition of ATPase activity.4.The functions of cadaverine transport protein CadB were studied in Escherichia colt: CadB had both cadaverine uptake and cadaverine excretion activities. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine, leading to generation of a proton motive force. The cadBA operon plays important roles in cellular regulation at acidic pH. Less