Search for polyamine agonists and antagonists acting on NMDA receptor.

寻找作用于NMDA受体的多胺激动剂和拮抗剂。

• 批准号: 07557376
• 负责人: IGARASHI Kazuei
• 金额: $ 1.02万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557376/
• 关键词: Polyamine; Spermine; NMDA receptor; Agonist; Antagonist; Channel blocker; ポリアンミン; ビスエチルポリアミン

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 PotD,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 mutated … More . This region of NR1A shows homology with bacterial amono acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine (D669A), or glutamate (D669E) abolished sperimine stimulation. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutation 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. 为了确定精胺结合的重要氨基酸残基，我们使用定点诱变技术改变NMDA受体NR1亚基区域内和周围的氨基酸，该区域与大肠杆菌中的多胺结合蛋白PotD具有同源性。用电压箝法研究了异质和同源NMDA受体突变亚基在非洲爪蟾卵母细胞中的表达。两个酸性残基（E339或E342）突变为中性氨基酸，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伊芬丙地尔的敏感性也有一定的影响，但对谷氨酸和甘氨酸或Mg^<2+>的电压依赖性阻断的敏感性没有改变。NR1中的残基E342似乎对精胺刺激至关重要。接下来，位于NR1A亚基假定的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。NR1A的这一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），消除了对精子的刺激。这些突变还显著降低了伊芬普罗地尔和质子对NR1A/NR2B受体的抑制作用。NR1A （D669）突变对谷氨酸和甘氨酸的效力几乎没有影响，也不会改变Mg^<2+>或精胺刺激的“甘氨酸依赖”形式的电压依赖性阻断。令人惊讶的是，D669N和D669A突变，而不是D669E突变，减少了精胺的电压依赖性阻滞。NR1A中的D669可以构成多胺和伊芬普罗地尔结合位点的一部分和/或NMDA受体的质子传感器的一部分。研究了N^1-丹酰精胺（N^1- danspm）和N^1-（正辛烷磺酰基）精胺（N^1- osspm）对爪蟾卵母细胞中表达的重组NMDA受体的影响。在-70 mv电压下，N^1-DanSpm和N^1-OsSpm抑制NMDA受体，比精胺在卵母细胞中的作用强1000倍。N^1-DanSpm和N^1-OsSpm的阻滞具有很强的电压依赖性，在超极化膜电位处更为明显。少

项目成果

Igarashi, K., and Williams, K.: "Antagonist properties of polyamines and bis (ethyl) polyamines at N-methyl-D-aspartate receptors." J.Pharmacol.Exp.Ther.272. 1101-1109 (1995)

Igarashi, K. 和 Williams, K.：“多胺和双（乙基）多胺对 N-甲基-D-天冬氨酸受体的拮抗特性。”

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。”

K. Williams et al.: "An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation." Mol. Pharmacol.48. 1087-1098 (1995)

K. Williams 等人：“N-甲基-D-天冬氨酸受体中的一种酸性氨基酸，对精胺刺激很重要。”

K.Williams et al.: "Activation of N-methy1-D-aspartate receptors by glycine : Role of an aspartate residue in the M3-M4 loop of the NR1 subunit." Mol. Pharmacol.50. 701-708 (1996)

K.Williams 等人：“甘氨酸激活 N-甲基1-D-天冬氨酸受体：NR1 亚基 M3-M4 环中天冬氨酸残基的作用。”