Regulation of NMDA receptor by polyamine and its derivatives

多胺及其衍生物对NMDA受体的调节

• 批准号: 08044249
• 负责人: IGARASHI Kazuei
• 金额: $ 1.41万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044249/
• 关键词: Polyamine; Spermine; NMDA receptor; Antagonist; Site-directed mutagenesis; 部位突然変異

1. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with pot D,a Polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339 or E342) to neutral amino acids reduced or abolished stimulation by spermine without affecting voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg^<2+>. Residue E342 in NR1 appears to be critical for spermine stimulation. Next, sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutate … More d. This region of NR1A shows homology with bacterial amino acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine(D669A), orglutamate (D669E) abolished spermine stimulation. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg^<2+> or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor.2. The effects of several N-sulfonyl-polyamines, including N^1-dansyl-spermine (N^1-DanSpm) and N^1-(n-octanesulfonyl)-spermine (N^1-OsSpm), were studied at recombinant NMDA receptors expressed in Xenopus oocytes. N^1-DanSpm and N^1-OsSpm inhibited NMDA receptors and were about 1,000-fold more potent than spermine in oocytes voltage-clamped at -70 mV.Block by N^1-DanSpm and N^1-OsSpm was strongly voltage-dependent, being more pronounced at hyperpolarized membrane potentials. Less

1。确定氨基酸保留对精子结合至关重要的氨基酸，我们使用定位定向的诱变来改变NMDA受体NR1亚基的区域内及其周围的氨基酸，该区域显示了与pot d（一种来自Escherichia coli的多胺结合蛋白）的同源性。在异源和同源性NMDA受体中表达的突变亚基通过电压钳记录在Xenopus卵母细胞中。对中性氨基酸的两个酸性保留（E339或E342）的突变减少或废除了精子的刺激，而不会影响精子的电压依赖性块。这些残留物的突变也对质子和ifenprodil的敏感性有适度的影响，但并未改变对谷氨酸和甘氨酸的敏感性，或者通过mg^<2+>对电压依赖性阻滞。 NR1中的残基E342似乎对于精子刺激至关重要。接下来，位于NR1A亚基的假定细胞外环的前两个三分之二的16个谷氨酸和天冬氨酸保留。 NR1A的该区域与细菌多胺结合蛋白和细菌精子乙酰基转移酶的同源性。 D669至天冬氨酸（D669N），丙氨酸（D669A），奥格鲁略氨酸（D669E）的突变消除了精子刺激。这些突变还显着降低了Ifenprodil和NR1A/NR2B受体处质子的抑制作用。 NR1A的突变（D669）对谷氨酸和甘氨酸的电位几乎没有影响，并且不会通过Mg^<2+>或“甘氨酸依赖性”形式的精子刺激改变电压依赖性块。令人惊讶的是，D669N和D669A突变，但不是D669E突变，可通过精子降低电压依赖性块。 NR1A中的D669可以构成多胺和ifenprodil和/或NMDA受体的质子传感器的一部分的一部分。2。在重组NMDA受体下，在重组NMDA受体下研究了几种N-磺酰基聚酰胺，包括N^1-二硫代植物（N^1-DansPM）和N^1-（N- octanesulfonyl） - 胸膜（N^1-OSSPM）的影响。 N^1-dansPM和N^1-OSSPM抑制了NMDA受体，并且在-70 mV.block中，卵母细胞在-70 mv.block中的卵母细胞的潜力高约1,000倍。较少的

项目成果

S.Sugiyama et al.: "The 1.8* X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding." Protein Science. 5. 1984-1990 (1996)

S.Sugiyama 等人：“大肠杆菌 PotD 蛋白与亚精胺复合的 1.8* X 射线结构以及多胺结合机制。”

S.Sugiyama et al.: "The 1.8 Å X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding." Protein Science. 5. 1984-1990 (1996)

S.Sugiyama 等人：“大肠杆菌 PotD 蛋白与亚精胺复合的 1.8 Å X 射线结构以及多胺结合的机制。” 5. 1984-1990 (1996)。

J.Chao et al.: "N^1-Dansylspermine and N^1-(n-octanesulfonyl)-spermine, novel glutamate receptor antagonists : Block and permeation of N-methyl-D-aspartate receptors." Mol.Pharmacol.51 (in press). (1996)

J.Chao 等人：“N^1-Dansylspermine 和 N^1-(n-octanesulfonyl)-spermine，新型谷氨酸受体拮抗剂：阻断和渗透 N-甲基-D-天冬氨酸受体。”

S.Sugiyama et al.: "Crystal structure of PotD,the primary receptor of the polyamine transport system in Escherichia coli." J.Biol.Chem.271. 9519-9525 (1996)

S.Sugiyama 等人：“PotD 的晶体结构，大肠杆菌中多胺转运系统的主要受体。”

K.Kashiwagi et al.: "An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons." Mol.Pharmacol.49. 1131-1141 (1996)

K.Kashiwagi 等人：“N-甲基-D-天冬氨酸受体细胞外环中的天冬氨酸残基控制着对精胺和质子的敏感性。”