Regulation of glutamate receptors by polyamines

1.我们先前已经发现，NR1亚基上的几个酸性残基影响对精胺和质子的敏感性。为了寻找更多的残基，我们利用电压钳记录在X enopus卵母细胞中表达的NR1/NR2B受体，研究了NR1亚基中所有胞外酸性残基的突变。外显子5插入序列附近的E181和E185残基以及一些下游残基的突变减少了精胺的刺激和质子抑制，而不影响精胺的电压依赖性阻断。在某些情况下，这些突变体的影响是相加的。例如，一个双NR1(E181Q，E185Q)突变体比每个单独突变体有更大的影响。一些酸性残基可能有助于与精胺或质子的结合位点，而另一些可能与通道门结合。应用重组酶…电压钳记录技术研究氨基糖苷类抗生素对N-甲基-D-天冬氨酸受体的影响X enopus卵母细胞表达较多的NT-NMDA受体。一些氨基糖苷类抗生素可增强异构体NR1A/NR2B受体的宏观电流，但不能增强NR1A/NR2A、NR1A/NR2C、NR1A/NR2D和NR1B/NR2B受体的宏观电流。200亩M抗生素的增效作用大小顺序为：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。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。这些结果提示氨基糖苷类化合物具有刺激作用，这种刺激作用是通过与NMDA受体上精胺结合部位的结合来实现的。在-70 mV电压钳制下，一些末端或中心氨基上有苄基取代的单、二和三苄基多胺可抑制卵母细胞NR1/NR2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IC50和GT；值为0.2muM.TB-3-4具有强烈的电压依赖性。在10 mM时，TB-3-4对ci-amino-3-hydroxy-5-methyl-4-isoxazolepropionic亚单位表达的NMDAICD型受体无影响，提示TB-3-4是一种选择性NMDA型拮抗剂。较少

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