Durand G M, Gregor P, Zheng X, Bennett M V, Uhl G R, Zukin R S
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461.
Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9359-63. doi: 10.1073/pnas.89.19.9359.
Molecular cloning identified complementary DNA species, from a rat ventral midbrain library, encoding apparent splice variants of the N-methyl-D-aspartate (NMDA) receptor NMDAR1 (which we now term NR1a). Sequencing revealed that one variant, NR1b, differs from NR1a by the presence of a 21-amino acid insert near the amino end of the N-terminal domain and by an alternate C-terminal domain in which the last 75 amino acids are replaced by an unrelated sequence of 22 amino acids. NR1b is virtually identical to NR1a in the remainder of the N- and C-terminal domains, at the 5' and 3' noncoding ends, and within the predicted transmembrane domains and extracellular and cytoplasmic loops. These findings suggest that the two forms of the receptor arise by differential splicing of a transcript from the same gene. Sequencing of other clones indicates the existence of a third variant, NR1c, identical to NR1b in its C terminus but lacking the N-terminal insert. NR1b RNA injected into Xenopus oocytes generated functional homomeric NMDA channels with electrophysiological properties distinct from those of NR1a homomeric channels. NR1b channels exhibited a lower apparent affinity for NMDA and for glutamate. NR1b channels exhibited a lower affinity for D-2-amino-5-phosphonovaleric acid and a higher affinity for Zn2+. The two receptor variants showed nearly identical affinities for glycine, Mg2+, and phencyclidine. Spermine potentiation of NMDA responses, prominent in oocytes injected with rat forebrain message, was also prominent for NR1a receptors, but was greatly reduced or absent for NR1b receptors. Treatment with the protein kinase C activator phorbol 12-myristate 13-acetate potentiated NMDA responses in NR1b-injected oocytes by about 20-fold; potentiation of NMDA responses in NR1a-injected oocytes was much less, about 4-fold. These findings support a role for alternate splicing in generating NMDA channels with different functional properties.
分子克隆从大鼠腹侧中脑文库中鉴定出互补DNA物种，其编码N-甲基-D-天冬氨酸（NMDA）受体NMDAR1（我们现在称之为NR1a）的明显剪接变体。测序显示，一种变体NR1b与NR1a的不同之处在于，在N端结构域的氨基末端附近存在一个21个氨基酸的插入片段，以及一个不同的C端结构域，其中最后75个氨基酸被22个氨基酸的不相关序列所取代。NR1b在N端和C端结构域的其余部分、5'和3'非编码末端以及预测的跨膜结构域和细胞外及细胞质环内与NR1a几乎相同。这些发现表明，受体的两种形式是由同一基因转录本的差异剪接产生的。对其他克隆的测序表明存在第三种变体NR1c，其C末端与NR1b相同，但缺少N端插入片段。注射到非洲爪蟾卵母细胞中的NR1b RNA产生了功能性同聚体NMDA通道，其电生理特性与NR1a同聚体通道不同。NR1b通道对NMDA和谷氨酸的表观亲和力较低。NR1b通道对D-2-氨基-5-膦酰基戊酸的亲和力较低，对Zn2+的亲和力较高。这两种受体变体对甘氨酸、Mg2+和苯环己哌啶的亲和力几乎相同。在注射大鼠前脑信息的卵母细胞中显著的精胺对NMDA反应的增强作用，对NR1a受体也很显著，但对NR1b受体则大大降低或不存在。用蛋白激酶C激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯处理可使注射NR1b的卵母细胞中的NMDA反应增强约20倍；注射NR1a的卵母细胞中NMDA反应的增强作用要小得多，约为4倍。这些发现支持了可变剪接在产生具有不同功能特性的NMDA通道中的作用。
