Regulation of glutamate receptors by polyamines

多胺对谷氨酸受体的调节

• 批准号: 09044259
• 负责人: IGARASHI Kazuei
• 金额: $ 2.75万
• 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: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044259/
• 关键词: Polyamine; Spermine; NMDA receptor; Binding site; Site-directed mutagenesis; Mg^<2+>; Aminoglycoside; Channel blocker; 活性調節; アンタゴニスト

1. We have previously found that several acidic residues in the NR1 subunit influence sensitivity to spermine and protons. To look for additional residues that are involved, we studied mutations at all of the extracellular acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in X enopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, and at some downstream residues, reduced spermine stimulation and proton inhibition without affecting voltage-dependent block by spermine. In some cases the effects of these mutants were additive. For example, a double NR1 (E181Q, E185Q) mutant had a larger -effect than each individual mutant. Some of the acidic residues may contribute to binding sites for spermine or protons, and others may act to couple binding to channel gating.2. The effects of aminoglycoside antibiotics on N-methyl-D-aspartate (NMDA) receptors were studied using voltage-clamp recording of recombina … More nt NMDA receptors expressed in X enopus oocytes. A number of aminoglycoside antibiotics were found to potentiate macroscopic currents at heteromeric NR1A/NR2B receptors, but not at NR1A/NR2A, NR1A/NR2C, NR1A/NR2D and NR1B/NR2B receptors. The dugree of potentiation with 200 mu M antibiotic had a rank order neomycin B > paromomycin > gentamicin C > geneticin > kanamycin A > streptomycin. Potentiation was not seen with kasugamycin and spectinomycin. The degree of stimulation paralleled the number of the amino groups in the aminoglycosides. We measured the effects of aminoglycosides at mutant NMDA receptors to determine which amino acid residues in NMDA receptor subunits are involved in stimulation. Mutations that reduced or abolished spermine stimulation also reduced stimulation by aminoglycosides. The results suggest that aminoglycosides have stimulatory effects that are mediated through binding to the spermine binding site on NMDA receptors.3. A number of mono-, di- and tri-benzyl polyamines, having benzyl substitutions on the terminal or central amino groups, inhibited responses of NR1/NR2 receptors in oocytes voltage-clamped at -70 mV.Among the most potent compouncds was N^1, N^4, N^8 -tri-benzyl-spermidine (PB-3-4), which had an IC_<50> value of 0.2 muM.TB-3-4 was strongly voltage dependent. At a concentration of 10 muM, TB-3-4 had no effect on ci-amino-3-hydroxy-5-methyl-4-isoxazolepropionic aicd receptors expressed from the GluR1 subunit, indicating that TB-3-4 is a selective NMDA antagonist. Less

1.我们以前已经发现，NR 1亚基中的几个酸性残基影响精胺和质子的敏感性。为了寻找更多的残基参与，我们研究了突变的所有细胞外的酸性残基在NR 1亚基使用电压钳记录NR 1/NR 2B受体在X enopus卵母细胞中表达。在外显子5插入位点附近的残基突变，包括E181和E185，以及在一些下游残基，减少精胺刺激和质子抑制，而不影响电压依赖性阻断精胺。在某些情况下，这些突变体的影响是累加的。例如，双NR 1（E181 Q，E185 Q）突变体比每个单独的突变体具有更大的-效应。一些酸性残基可能有助于精胺或质子的结合位点，而另一些则可能起到将结合与通道门控偶联的作用。应用电压钳技术研究了氨基糖苷类抗生素对N-甲基-D-天冬氨酸（NMDA）受体的作用。 ...更多信息 nt NMDA受体在非洲爪蟾卵母细胞中表达。发现许多氨基糖苷类抗生素增强异聚体NR 1A/NR 2B受体的宏观电流，但不增强NR 1A/NR 2A、NR 1A/NR 2C、NR 1A/NR 2D和NR 1B/NR 2B受体的宏观电流。200 μ M抗生素的增效作用大小依次为新霉素B >巴龙霉素>庆大霉素C >遗传霉素>卡那霉素A >链霉素。春雷霉素和大观霉素未观察到增效作用。刺激的程度决定了氨基糖苷类中氨基的数量。我们测量了氨基糖苷类对突变型NMDA受体的作用，以确定NMDA受体亚基中哪些氨基酸残基参与刺激。减少或消除精胺刺激的突变也减少了氨基糖苷类的刺激。结果提示，氨基糖苷类药物通过与NMDA受体上的精胺结合位点结合而发挥兴奋作用.在-70 mV电压钳位条件下，许多末端或中心氨基上有苄基取代的单苄基、二苄基和三苄基多胺抑制卵母细胞NR 1/NR 2受体的反应。其中最有效的化合物是N^1，N^4，N^8 -三苄基亚精胺（PB-3-4），其IC_<50>值为0.2 μ M。TB-3-4具有强烈的电压依赖性。在10 μ M浓度下，TB-3-4对由GluR 1亚基表达的顺-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体没有作用，表明TB-3-4是选择性NMDA拮抗剂。少

项目成果

T. Fukuchi-Shimogari et al.: "Malignant transformation by overproduction of translation initiation factor eIF4G." Cancer Res.57. 5041-5044 (1997)

T. Fukuchi-Shimogari 等人：“翻译起始因子 eIF4G 过量产生的恶性转化。”

K. Igarashi et al.: "Molecular mechanism of polyamines stimukation of the synthesis of oligopeptide binding protein." J. Biol. Chem.272. 4058-4064 (1997)

K. Igarashi 等人：“多胺刺激寡肽结合蛋白合成的分子机制。”

K.Kashiwagi et al.: "Block and modulation of N-methyl-D-aspartate receptors by polyamines and protons : Role of amino acid residues in the transmembrane and pore-forming regions of NR1 and NR2 subunits." Mol. Pharmacol.52. 701-703 (1997)

K.Kashiwagi 等人：“多胺和质子对 N-甲基-D-天冬氨酸受体的阻断和调节：氨基酸残基在 NR1 和 NR2 亚基的跨膜和孔形成区域中的作用。”

K.Kashiwagi et al.: "Block and modulation of N-methyl-D-aspartate receptors by polyamines and protons : Role of amino acid residues in the transmembrane and pore-forming regions of NR1 and NR2 sububits." Mol.Pharmacol.52. 701-703 (1997)

K.Kashiwagi 等人：“多胺和质子对 N-甲基-D-天冬氨酸受体的阻断和调节：氨基酸残基在 NR1 和 NR2 亚基的跨膜和孔形成区域中的作用。”

K. Kashiwagi et al.: "Excretion and uptake of putrescine by the PotE protein in Escherichia coli." J. Biol. Chem.272. 6318-6323 (1997)

K. Kashiwagi 等人：“大肠杆菌中 PotE 蛋白对腐胺的排泄和吸收。”