An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons.
An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons.
复制标题
DOI：
--
复制
发表时间：
1996-06
影响因子：
3.6
通讯作者：
K. Kashiwagi;J. Fukuchi;J. Chao;K. Igarashi;K. Williams
中科院分区：
文献类型：
--
作者：
K. Kashiwagi;J. Fukuchi;J. Chao;K. Igarashi;K. Williams
为了研究酸性残基在精胺调节NMDA受体中的作用，我们采用了受体亚基定点突变和电压钳记录技术在非洲爪蟾卵母细胞中。16个谷氨酸和天冬氨酸残基，位于前三分之二的推定的细胞外环的NR 1A亚基，分别突变。NR 1A的这一区域显示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的同源性。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）可消除异聚体NR 1A/NR 2B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NR 1A/NR 2B受体的抑制作用。在NR 1B中的等同位置（D 690）的突变，其包含由外显子5编码的插入物，降低了NR 1B/NR 2B受体的pH敏感性。因此，外显子5的存在并不能阻止D 669突变的影响，外显子5的影响也不能阻止D 669突变（NR 1B中的D 690）。NR 1A（D 669）突变对谷氨酸和甘氨酸的效力影响很小或没有影响，并且不改变Mg 2+或精胺刺激的“甘氨酸依赖性”形式的电压依赖性阻断。令人惊讶的是，D 669 N和D 669 A突变，而不是D 669 E突变，减少了精胺对NR 1A/NR 2受体的电压依赖性阻滞。NR 2B中相当于D 669的位置（D 668）突变不会改变精胺刺激或对pH和艾芬地尔的敏感性。然而，NR 2B中的突变D 668 N和D 668 A而不是D 668 E减少精胺的电压依赖性阻滞。精胺对NR 1A（D 669）和NR 2B（D 668）负电荷的屏蔽作用可能与电压依赖性阻滞有关。NR 1A中的D 669可以形成多胺和艾芬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。或者，该残基对于调节剂如精胺、质子和艾芬地尔与通道门控的偶联可能是关键的。
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.