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。确定氨基酸保留对精子结合很重要的氨基酸，我们使用定位定向的诱变来改变NMDA受体NR1亚基的区域和周围的氨基酸，该区域显示与Escherichia coli的多胺结合蛋白POTD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）或谷氨酸（D669E）的突变消除了蜘蛛刺激。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，并且比在-70 mV时粘液的卵母细胞中的精子高约1,000倍。 n^1-danspm的块和n^1-osspm强烈依赖电压，在超极化膜电位上更为明显。较少的

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. 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。”