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.在N-甲基-D-天冬氨酸（NMDA）受体上，精子，质子和Ifenprodil之间存在复杂的相互作用。精子刺激可能涉及缓解质子抑制作用，而Ifenprodil抑制作用可能涉及质子抑制的增加。我们使用在异爪蟾卵母细胞中表达的NR1/NR2B受体的电压夹记录在NR1亚基中酸性保留时研究了突变。外显子5插入部位附近的救援突变（包括E181和E185）减少了精子刺激和质子抑制作用。突变NR1（D130N）使对Ifenprodil的敏感性降低了500倍以上，但对对精子和PH的敏感性几乎没有影响。在NR1亚基这一区域中，其他六个残留物在其他六个残留物中降低了效力，在某些情况下，最大的效应是ifenproploproprodiroploprodiroploprodiroploprodil的效力。这些突变体不影响对NMDA通道的pH，谷氨酸，甘氨酸或其他标志性能的敏感性，例如mg^<2+> blodk和Ba^<2+>渗透率。该地区的残留物可能是fo…更多的RM ifenprodil结合部位的一部分。为了模拟NR1的该区域，我们将NR1（残基19-400）的预测二级结构与已知结构的1,400蛋白质进行了比较。 NR1的该区域与细菌亮氨酸/异亮氨酸/丝线结合蛋白，这是一种包含两种爱的全球氨基酸结合蛋白，类似于谷氨酸受体的下游S1-S2区域。我们建议NR1（22-375）的三级结构类似于亮氨酸/异亮氨酸/瓣膜结合蛋白，其中包含两个“调节”结构域，我们将其称为R1和R2。该区域包含精子和Ifenprodil的结合位点，可能会使用氨基糖苷抗生素对NMDA受体的影响，可能会影响下游S1和S2结构域，使用电压夹记录NMDA受体的电压杆会记录Xenopus in Xenopus ocyyopus ocyyopus ocyyopus。发现许多氨基糖苷在异源NR1_A/NR2B受体上可能具有宏观电流，但在NR1_A/NR2A，NR1_A/NR2C，NR1_A/NR1_A/NR2D或NR1_B/NR1_B/NR2B受体。潜在的程度具有等级序列新霉素B>降低霉素>庆大霉素C>遗传素>卡纳米霉素A>链霉素。 kasugamycin和spectinomycin未见增强。刺激程度与氨基糖苷中氨基的数量平行。氨基糖苷的刺激作用在甘氨酸和酸性pH下的掺杂浓度更为明显，类似于精子的刺激作用。我们测量了突变NMDA受体对氨基糖苷的作用，以确定哪些氨基酸在NMDA受体亚基中保留的氨基酸涉及刺激。减少或废除精子刺激的突变也减少了氨基糖苷的刺激。几种氨基糖苷产生了NMDA受体的弱电压依赖性块，但抑制程度似乎与分子中氨基糖苷的数量没有相关。结果表明，氨基糖苷具有三个以上的氨基糖苷具有刺激作用，这些作用是通过NMDA受体上的精子结合位点介导的。3。发现蒽醌的多胺衍生物是NMDA受体拮抗剂。较少的

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)

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/

Tomitori, H.et al.: "Multiple polyamine transport systems on the vacuolar membrane in yeast."Biochem.J.. 353. 681-688 (2001)

Tomitori, H.et al.：“酵母液泡膜上的多重多胺转运系统。”Biochem.J.. 353. 681-688 (2001)

H.Tomitori et al.: "Multiple polyamine transport systems on the vacuolar membrane in yeast."Biochem.J.. 353. 681-688 (2001)

H.Tomitori 等人：“酵母液泡膜上的多重多胺转运系统。”Biochem.J. 353. 681-688 (2001)

Masuko, T.et al: "Stimulatory and inhibitory properties of aminoglycoside antibiotics at N-methyl-D-aspartate receptors."J.Pharmacol.Exp.Ther.. 290. 1026-1033 (1999)

Masuko, T.等人：“氨基糖苷类抗生素对 N-甲基-D-天冬氨酸受体的刺激和抑制特性。”J.Pharmacol.Exp.Ther.. 290. 1026-1033 (1999)