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. 为了鉴定对精胺结合重要的氨基酸残基，我们使用定点诱变来改变 NMDA 受体 NR1 亚基区域内和周围的氨基酸，该区域与大肠杆菌的多胺结合蛋白 pot D 同源。通过爪蟾卵母细胞中的电压钳记录研究了在异聚和同聚 NMDA 受体中表达的突变亚基。两个酸性残基（E339或E342）突变为中性氨基酸会减少或消除精胺的刺激，而不影响精胺的电压依赖性阻断。这些残基的突变对质子和艾芬地尔的敏感性也具有适度的影响，但没有改变对谷氨酸和甘氨酸或Mg 2+ 的电压依赖性阻断的敏感性。 NR1 中的 E342 残基似乎对于精胺刺激至关重要。接下来，位于 NR1A 亚基假定细胞外环的前三分之二的 16 个谷氨酸和天冬氨酸残基分别发生突变……更多 d。 NR1A 的该区域与细菌氨基酸结合蛋白、细菌多胺结合蛋白和细菌亚精胺乙酰转移酶具有同源性。 D669 突变为天冬酰胺 (D669N)、丙氨酸 (D669A) 或谷氨酸 (D669E) 消除了精胺刺激。这些突变还显着降低了艾芬地尔和质子对 NR1A/NR2B 受体的抑制作用。 NR1A(D669)的突变对谷氨酸和甘氨酸的效力影响很小或没有影响，并且不改变Mg 2+ 的电压依赖性阻断或精胺刺激的“甘氨酸依赖性”形式。令人惊讶的是，D669N 和 D669A 突变（而非 D669E 突变）减少了精胺的电压依赖性阻断。 NR1A中的D669可以形成多胺和艾芬地尔的结合位点的一部分和/或NMDA受体的质子传感器的一部分。2．研究了几种 N-磺酰基多胺，包括 N^1-丹磺酰基精胺 (N^1-DanSpm) 和 N^1-(正辛磺酰基)-精胺 (N^1-OsSpm) 对非洲爪蟾卵母细胞中表达的重组 NMDA 受体的影响。 N^1-DanSpm 和 N^1-OsSpm 抑制 NMDA 受体，在电压钳位于 -70 mV 的卵母细胞中，其效力比精胺强约 1,000 倍。N^1-DanSpm 和 N^1-OsSpm 的阻断具有强烈的电压依赖性，在超极化膜电位下更为明显。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。”

K.Kashiwagi et al.: "Spermidine-preferential uptake system in Escherichia coli.Identifi-cation of amino acids involved in polyamine binding in PotD protein." J.Biol.Chem.271. 12205-12208 (1996)

K.Kashiwagi 等人：“大肠杆菌中的亚精胺优先摄取系统。PotD 蛋白中参与多胺结合的氨基酸的鉴定。”

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。”