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

1.精胺、质子与N-甲基-D-天冬氨酸(NMDA)受体之间存在复杂的相互作用。精胺刺激可能涉及解除质子抑制，而异丙苯地尔抑制可能涉及质子抑制的增加。我们利用电压钳记录技术研究了非洲爪哇卵母细胞中表达的NR1/NR2B受体的酸性残基突变。外显子5插入物附近残基的突变，包括E181和E185，减少了精胺刺激和质子抑制。突变NR1(D130N)使患者对异丙苯地尔的敏感性降低500倍以上，但对精胺和苯丙氨酸的敏感性几乎没有影响。NR1亚基该区域其他6个残基的突变降低了异丙苯地尔的药效，在某些情况下降低了最大效应。这些突变体不影响NMDA通道对pH、谷氨酸、甘氨酸的敏感性，也不影响NMDA通道的其他特征特性，如镁离子通透性和Ba2+通透性。该区域的残基可能是…的残基更多的是依芬地尔结合部位的Rm部分。为了对NR1的这个区域进行建模，我们将预测的NR1二级结构(残基19-400)与1,400个蛋白质的已知结构进行了比较。NR1的这一区域与细菌亮氨酸/异亮氨酸/缬氨酸结合蛋白最为相似，后者是一种含有两个叶的球形氨基酸结合蛋白，类似于谷氨酸受体下游的S1-S2区域。我们认为NR1(22-375)的三级结构类似于亮氨酸/异亮氨酸/缬氨酸结合蛋白，包含两个“调节”结构域，我们称之为R1和R2。该区域包含精胺和异丙苯地尔的结合部位，可能影响构成甘氨酸结合口袋的下游S1和S2结构域。2.通过电压钳记录非洲爪哇卵母细胞表达的重组NMDA受体，研究氨基糖苷类抗生素对NMDA受体的影响。许多氨基糖苷类药物可增强NR1A/NR2B异构体受体的宏观电流，但对NR1A/NR2A、NR1A/NR2C、NR1A/NR2D或NR1B/NR2B受体无明显作用。增强程度由大到小依次为：新霉素B&gt；帕罗霉素&gt；庆大霉素C&gt；遗传素&gt；卡那霉素A&gt；链霉素。卡苏霉素和壮观霉素未见明显的增强作用。刺激程度与氨基糖苷类化合物中氨基的数量成正比。氨基糖苷类药物的刺激作用在甘氨酸亚饱和浓度和酸性pH条件下更为明显，与精胺的刺激作用相似。我们测量了氨基糖苷类化合物对突变型NMDA受体的影响，以确定NMDA受体亚基中哪些氨基酸残基参与了刺激。减少或取消精胺刺激的突变也会减少氨基糖苷类药物的刺激。几种氨基糖苷类药物对NMDA受体产生微弱的电压依赖性阻断，但抑制程度似乎与分子中氨基的数量无关。这些结果表明，含有三个以上氨基的氨基糖苷类化合物具有刺激作用，这种刺激作用是通过NMDA受体上的精胺结合部位来实现的。研究发现，多胺类化合物是一种NMDA受体拮抗剂。较少

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