Polyamines as biomodulators - regulation of their contents and their physiological functions

多胺作为生物调节剂 - 其含量及其生理功能的调节

• 批准号: 11694246
• 负责人: IGARASHI Kazuei
• 金额: $ 2.88万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694246/
• 关键词: Polyamine; Spermine; NMDA receptor; Regulatory domain; Aminoglycoside; Ototoxicity; Channel blocker; Anthraquinon derivative; イフェンプロジル

1.There are complex interactions between spermine, protons, and ifenprodil at N-methyl-D-aspartate(NMDA)receptors. Spermine stimulation may involve relief of proton inhibition, whereas ifenprodil inhibition may involve an increase in proton inhibition. We studied mutations at acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in Xenopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, reduced spermine stimulation and proton inhibition. Mutation NR1(D130N)reduced sensitivity to ifenprodil by more than 500-fold, but had little effect on sensitivity to spermine and pH.Mutations at six other residues in this region of the NR1 subunit reduced the potency and, in some cases, the maximum effect of ifenprodil. These mutants did not affect sensitivity to pH, glutamate, glycine, or other hallmark properties of NMDA channels such as Mg^<2+> blodk and Ba^<2+> permeability. Residues in this region presumably fo … More rm part of the ifenprodil-binding site. To model this region of NR1 we compared the predicted secondary structure of NR1(residues 19-400)with the known structures of 1,400 proteins. This region of NR1 is most similar to bacterial leucine/isoleucine/valine binding protein, a globular amino acid binding protein containing two lobes, similar to the downstream S1-S2 region of glutamate receptors. We propose that the tertiary structure of NR1(22-375)is similar to leucine/isoleucine/valine binding protein, containing two "regulatory" domains, which we term R1 and R2. This region, which contains the binding sites for spermine and ifenprodil, may influence the downstream S1 and S2 domains that constitute the glycine binding pocket.2.The effects of aminoglycoside antibiotics on NMDA receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. A number of aminoglycosides were found to potentiate macroscopic currents at heteromeric NR1_A/NR2B receptors, but not at NR1_A/NR2A, NR1_A/NR2C, NR1_A/NR2D or NR1_B/NR2B receptors. The degree of potentiation 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. The stimulatory effects of aminoglycosides were more pronounced at subsaturating concentrations of glycine and at acidic pH, similar to the stimulatory effects of spermine. 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. Several aminoglycosides produced a weak voltage-dependent block of NMDA receptors, but the degree of inhibition did not appear to correlated with the number of amino groups in the molecule. The results suggest that aminoglycosides having more than three amino groups have stimulatory effects that are mediated through the spermine-binding site on NMDA receptors.3. Polyamine derivatives of anthraquinon were found to be NMDA receptor antagonists. Less

1.精胺、质子和艾芬地尔在N-甲基-D-天冬氨酸（NMDA）受体上存在复杂的相互作用。精胺刺激可能涉及质子抑制的缓解，而艾芬地尔抑制可能涉及质子抑制的增加。我们研究了突变的NR 1亚基中的酸性残基使用电压钳记录的NR 1/NR 2B受体在非洲爪蟾卵母细胞中表达。在外显子5插入位点附近的残基突变，包括E181和E185，减少精胺刺激和质子抑制。突变NR 1（D130 N）降低艾芬地尔的敏感性超过500倍，但对精胺和pH值的敏感性几乎没有影响。在该区域的NR 1亚基的其他六个残基的突变降低了效力，在某些情况下，艾芬地尔的最大效果。这些突变体不影响对pH、谷氨酸、甘氨酸的敏感性，也不影响NMDA通道的其他标志性特性，如Mg^2+ blodk和Ba^2+渗透性。该区域的残留物可能是 ...更多信息 rm部分的艾芬地尔结合位点。为了模拟NR 1的这一区域，我们将预测的NR 1二级结构（残基19-400）与1,400种蛋白质的已知结构进行了比较。NR 1的这一区域与细菌亮氨酸/异亮氨酸/缬氨酸结合蛋白最相似，这是一种含有两个叶的球状氨基酸结合蛋白，类似于谷氨酸受体的下游S1-S2区域。我们认为NR 1（22-375）的三级结构类似于亮氨酸/异亮氨酸/缬氨酸结合蛋白，含有两个“调节”结构域，我们称之为R1和R2。该区域含有精胺和艾芬地尔的结合位点，可能影响下游的S1和S2结构域，从而构成甘氨酸结合口袋。2.利用电压钳技术研究了氨基糖苷类抗生素对爪蟾卵母细胞表达的重组NMDA受体的影响。发现许多氨基糖苷类在异聚体NR1_A/NR 2B受体上增强宏观电流，但在NR1_A/NR 2A、NR1_A/NR 2C、NR1_A/NR 2D或NR1_B/NR 2B受体上不增强宏观电流。增效作用的强弱顺序为新霉素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。氨基糖苷类的刺激作用在甘氨酸的亚饱和浓度和酸性pH下更明显，类似于精胺的刺激作用。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。几种氨基糖苷类药物对NMDA受体产生弱的电压依赖性阻滞，但抑制程度似乎与分子中氨基的数量无关。结果表明，氨基多于3个的氨基糖苷类化合物具有兴奋NMDA受体的作用，这种作用是通过NMDA受体上的精胺结合位点介导的.蒽醌的多胺衍生物被发现是NMDA受体拮抗剂。少

项目成果

K.Igarashi and K.Kashiwagi: "Polyamines : Mysterious modulators of cellular functions. (Review)"Biochem.Biophys.Res.Commun.. 271. 559-564 (2000)

K.Igarashi 和 K.Kashiwagi：“多胺：细胞功能的神秘调节剂。（评论）”Biochem.Biophys.Res.Commun.. 271. 559-564 (2000)

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Yoshida,M, et al.：“多胺在翻译水平上增强腺苷酸环化酶的合成，从而刺激 RNA 聚合酶 σ^<2R> 亚基的合成。”J.Biol.Chem.. 276（in (2001)

Yoshida, M.et al.: "Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase σ^<28> subunit."J.Biol.Chem.. 276(in press). (2001)

Yoshida, M.等人：“多胺在翻译水平上增强腺苷酸环化酶的合成，从而刺激 RNA 聚合酶 σ^<28> 亚基的合成。”J.Biol.Chem.. 276（in (2001)

K.Kashiwagi et al.: "Identification of the putrescine recognition site on polyamine transport protein PotE."J.Biol.Chem.. 275. 36007-36012 (2000)

K.Kashiwagi 等：“多胺转运蛋白 PotE 上腐胺识别位点的鉴定。”J.Biol.Chem.. 275. 36007-36012 (2000)

Masuko, T.et al.: "A regulatory domain(R1-R2)in the amino terminus of the N-methyl-D-aspartate receptor : Effects of spermine, protons, and ifenprodil, and structural similarity to bacterial leucine/isoleucine/valine binding protein."Mol.Pharmacol.. 55. 9

Masuko, T.等人：“N-甲基-D-天冬氨酸受体氨基末端的调节域（R1-R2）：精胺、质子和艾芬地尔的作用，以及与细菌亮氨酸/异亮氨酸的结构相似性/