Search for polyamine agonists and antagonists acting on NMDA receptor.

1.为了确定对精胺结合很重要的氨基酸残基，我们使用定点突变技术改变了NMDA受体NR1亚单位内及其周围的氨基酸，该区域与来自大肠杆菌的多胺结合蛋白PotD同源。用电压钳记录技术研究了非洲爪哇卵母细胞中异构体和同构体NMDA受体突变亚基的表达。将两个酸性残基(E339或E342)突变为中性氨基酸可减少或取消精胺的刺激作用，而不影响精胺的电压依赖性阻断。这些残基的突变对质子和异丙苯地尔的敏感性也有轻微影响，但不改变对谷氨酸和甘氨酸的敏感性，也不改变对镁离子和2+&gt；电压依赖性阻断的敏感性。NR1中的E342残基似乎是精胺刺激的关键。接下来，位于NR1A亚单位胞外环的前三分之二的16个谷氨酸和天冬氨酸残基被分别突变为…更多。NR1a的这个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同源。D669突变为天冬酰胺(D669N)、丙氨酸(D669A)或谷氨酸(D669E)可抑制精胺刺激。这些突变还显著降低了异丙苯地尔和NR1A/NR2B受体上质子的抑制作用。NR1a(D669)突变对谷氨酸和甘氨酸的效价几乎没有影响，也不会改变镁离子或“甘氨酸依赖”形式的精胺刺激对电压依赖的阻断作用。令人惊讶的是，D669N和D669A突变，而不是D669E突变，减少了精胺对电压依赖的阻断。NR1a中的D669可以形成多胺和异丙苯地尔的结合位点的一部分和/或NMDA受体的质子传感器的一部分。研究了N^1-丹磺酰精胺(N^1-DanSpm)和N^1-(n-辛磺酰基)精胺(N^1-OsSpm)等N-磺酰多胺对非洲爪哇卵母细胞表达的重组NMDA受体的影响。N^1-DanSpm和N^1-OsSpm对NMDA受体均有抑制作用，在-70 mV电压钳制下的抑制作用约为精胺的1000倍。N^1-DanSpm和N^1-OsSpm的阻断作用具有强烈的电压依赖性，在超极化的膜电位下更为明显。较少

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