The NR1 M3 domain mediates allosteric coupling in the N-methyl-D-aspartate receptor
The NR1 M3 domain mediates allosteric coupling in the N-methyl-D-aspartate receptor
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DOI：
10.1124/mol.107.044115
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发表时间：
2008-08-01
影响因子：
3.6
通讯作者：
VanDongen, Antonius M. J.
中科院分区：
文献类型：
--
作者：
Blanke, Marie L.;VanDongen, Antonius M. J.
N-甲基-D-天冬氨酸(NMDA)受体在中枢神经系统发育和成人神经可塑性中发挥着重要作用。然而，尽管NMDA受体是一个有价值的治疗靶点，但其结构和功能特性之间的关系尚未完全阐明。为了进一步探索受体激活的机制，我们鉴定了NR1 M3片段中的两个功能获得突变，NR1 M3片段是一个连接配体结合和通道开放的跨膜域。两个突变体(A7Q和A7Y)都显示出显著的甘氨酸非依赖性电流，表明它们的M3结构域可能优先采用更活跃的构象。取代半胱氨酸修饰实验表明，A7Q和A7Y的甘氨酸结合裂隙都不能被修饰剂所接近，并能抵抗竞争性拮抗作用。这些数据表明，即使在没有激动剂的情况下，M3的扰动也可以稳定闭合裂隙构象中的配体结合域。两个突变体也表现出显著的谷氨酸非依赖性电流和对谷氨酸位点拮抗不敏感，这表明甘氨酸或谷氨酸单独激活。此外，A7Q和A7Y增加了NR2 M3结构域的可及性，为跨膜水平的亚基间偶联提供了证据，并表明这些NR1突变控制了完整的异构体受体的特性。NR2的等值突变没有表现出类似的表型，表明NR1和NR2的M3结构域可能发挥不同的功能作用。综上所述，我们的数据表明，NR1 M3片段在功能上与NR1和NR2亚基中的关键结构域相连。
N-Methyl-D-aspartate (NMDA) receptors play a critical role in both development of the central nervous system and adult neuroplasticity. However, although the NMDA receptor presents a valuable therapeutic target, the relationship between its structure and functional properties has yet to be fully elucidated. To further explore the mechanism of receptor activation, we characterized two gain-of-function mutations within the NR1 M3 segment, a transmembrane domain proposed to couple ligand binding and channel opening. Both mutants (A7Q and A7Y) displayed significant glycine-independent currents, indicating that their M3 domains may preferentially adopt a more activated conformation. Substituted cysteine modification experiments revealed that the glycine binding clefts of both A7Q and A7Y are inaccessible to modifying reagents and resistant to competitive antagonism. These data suggest that perturbation of M3 can stabilize the ligand binding domain in a closed cleft conformation, even in the absence of agonist. Both mutants also displayed significant glutamate-independent current and insensitivity to glutamate-site antagonism, indicating partial activation by either glycine or glutamate alone. Furthermore, A7Q and A7Y increased accessibility of the NR2 M3 domain, providing evidence for intersubunit coupling at the transmembrane level and suggesting that these NR1 mutations dominate the properties of the intact heteromeric receptor. The equivalent mutations in NR2 did not exhibit comparable phenotypes, indicating that the NR1 and NR2 M3 domains may play different functional roles. In summary, our data demonstrate that the NR1 M3 segment is functionally coupled to key structural domains in both the NR1 and NR2 subunits.