Kashiwagi K, Fukuchi J, Chao J, Igarashi K, Williams K
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, 19104-6084, USA.
Mol Pharmacol. 1996 Jun;49(6):1131-41.
To study the role of acidic residues in modulation of NMDA receptors by spermine, we used site-directed mutagenesis of receptor subunits and voltage-clamp recording in Xenopus oocytes. Sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutated. This region of NR1A shows homology with bacterial amino 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 the "glycine-independent" form of spermine stimulation in heteromeric NR1A/NR2B receptors. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at the equivalent position (D690) in NR1B, which contains the insert encoded by exon 5, reduced the pH sensitivity of NR1B/NR2B receptors. Thus, the effects of mutations at D669 are not prevented by the presence of exon 5, and the influence of exon 5 is not prevented by mutations at D669 (D690 in NR1B). Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg2+ 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 at NR1A/NR2 receptors. Mutations in NR2B at a position (D668) equivalent to D669 did not alter spermine stimulation or sensitivity to pH and ifenprodil. However, mutations D668N and D668A but not D668E in NR2B reduced voltage-dependent block by spermine. Screening of the negative charges at NR1A(D669) and NR2B(D668) may be involved in 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. Alternatively, this residue may be critical for coupling of modulators such as spermine, protons, and ifenprodil to channel gating.
为了研究酸性残基在精胺调节N-甲基-D-天冬氨酸（NMDA）受体中的作用，我们利用受体亚基的定点突变和非洲爪蟾卵母细胞中的电压钳记录技术进行了研究。位于NR1A亚基假定细胞外环前三分之二区域的16个谷氨酸和天冬氨酸残基被逐个突变。NR1A的这一区域与细菌氨基酸结合蛋白、一种细菌多胺结合蛋白以及一种细菌亚精胺乙酰转移酶具有同源性。将D669突变为天冬酰胺（D669N）、丙氨酸（D669A）或谷氨酸（D669E）消除了异聚体NR1A/NR2B受体中精胺刺激的“甘氨酸非依赖性”形式。这些突变还显著降低了艾芬地尔和质子对NR1A/NR2B受体的抑制作用。NR1B中相当于D669的位置（D690）发生突变，该位置包含由外显子５编码的插入片段，降低了NR1B/NR2B受体的pH敏感性。因此，D669处的突变效应不会因外显子５的存在而受到阻止，外显子５的影响也不会因D669（NR1B中的D690）处的突变而受到阻止。NR1A（D669）处的突变对谷氨酸和甘氨酸的效力几乎没有影响，也不会改变Mg2+引起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，D669N和D669A突变，但不是D669E突变，降低了NR1A/NR2受体中精胺引起的电压依赖性阻断。NR2B中与D669相当的位置（D668）发生突变，不会改变精胺刺激或对pH和艾芬地尔的敏感性。然而，NR2B中的D668N和D668A突变，但不是D668E突变，降低了精胺引起的电压依赖性阻断。对NR1A（D669）和NR2B（D668）处负电荷的筛选可能参与了精胺引起的电压依赖性阻断。NR1A中的D669可能构成多胺和艾芬地尔的结合位点的一部分和/或NMDA受体质子传感器的一部分。或者，该残基对于诸如精胺、质子和艾芬地尔等调节剂与通道门控之间的偶联可能至关重要。
