Search for polyamine agonists and antagonists acting on NMDA receptor.

1.为了确定对精胺结合重要的氨基酸残基，我们使用定点突变来改变NMDA受体NR 1亚基区域内和周围的氨基酸，该区域与大肠杆菌的多胺结合蛋白PotD具有同源性。突变的亚基，表达在异聚体和同源的NMDA受体，研究了电压钳记录在非洲爪蟾卵母细胞。两个酸性残基（E339或E342）突变为中性氨基酸减少或废除刺激精胺不影响电压依赖性阻断精胺。这些残基的突变对质子和艾芬地尔的敏感性也有适度的影响，但对谷氨酸和甘氨酸的敏感性或对Mg^<2+>的电压依赖性阻断的敏感性没有改变。NR 1中的残基E342似乎对精胺刺激至关重要。接下来，分别突变位于NR 1A亚基推定的细胞外环的前三分之二的16个谷氨酸和天冬氨酸残基 ...更多信息 . NR 1A的这一区域与细菌氨基酸结合蛋白、细菌多胺结合蛋白和细菌亚精胺乙酰转移酶具有同源性。将D 669突变为天冬酰胺（D 669 N）、丙氨酸（D 669 A）或谷氨酸（D 669 E）可消除精亚胺刺激。这些突变还显著降低了艾芬地尔和质子对NR 1A/NR 2B受体的抑制作用。NR 1A（D 669）突变对谷氨酸和甘氨酸的效力几乎没有影响，也没有改变Mg^<2+>的电压依赖性阻断或精胺刺激的“甘氨酸依赖性”形式。令人惊讶的是，D 669 N和D 669 A突变，而不是D 669 E突变，减少了精胺的电压依赖性阻滞。NR 1A中的D 669可能是多胺和艾芬地尔的结合位点的一部分，也可能是NMDA受体质子感受器的一部分.研究了几种N-磺酰多胺，包括N ^1-丹磺酰精胺（N^1-DanSpm）和N ^1-正辛烷磺酰精胺（N^1-OsSpm）对爪蟾卵母细胞表达的重组NMDA受体的作用。N^1-DanSpm和N^1-OsSpm抑制NMDA受体，并且在-70 mV电压钳位的卵母细胞中比精胺强约1，000倍。N^1-DanSpm和N^1-OsSpm的阻断具有强烈的电压依赖性，在超极化膜电位下更为明显。少

1. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD,a polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339 or E342) to neutral amino acids reduced or abolished stimulation by spermine without affecting voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg^<2+>. Residue E342 in NR1 appears to be critical for spermine stimulation. Next, sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutated … More . This region of NR1A shows homology with bacterial amono acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine (D669A), or glutamate (D669E) abolished sperimine stimulation. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutation at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg^<2+> or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor.2. The effects of several N-sulfonyl-polyamines, including N^1-dansyl-spermine (N^1-DanSpm) and N^1- (n-octanesulfonyl)-spermine (N^1-OsSpm), were studied at recombinant NMDA receptors expressed in Xenopus oocytes. N^1-DanSpm and N^1-OsSpm inhibited NMDA receptors and were about 1,000-fold more potent than spermine in oocytes voltage-clamped at -70 mv. Block by N^1-DanSpm and N^1-OsSpm was strongly voltage-dependent, being more pronounced at hyperpolarized membrane potentials. Less