The activation gate and gating mechanism of the NMDA receptor
The activation gate and gating mechanism of the NMDA receptor
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DOI：
10.1523/jneurosci.3485-07.2008
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发表时间：
2008-02-13
影响因子：
5.3
通讯作者：
Kuo, Chung-Chin
中科院分区：
文献类型：
--
作者：
Chang, Huai-Ren;Kuo, Chung-Chin
NMDA受体打开响应于NMDA和甘氨酸的结合。然而，目前尚不清楚NMDA受体孔的门控在哪里以及如何完成。我们发现NR2B中M3c的S645和I655之间的不同点突变（因此包括高度保守的SYTANLAAF基序）导致本构性开放通道。通过这些本构开放通道的电流很容易被外部Mg2+和MK-801[(+)-5-甲基-10,11-二氢- 5h -二苯并[a，d]环庚烯-5,10-马来酸亚胺]阻断。此外，当NMDA和甘氨酸被洗掉时，开放通道阻滞剂MK-801不能再被困在这些通道中。此外，A651或以下的M3c残基（NR2B， SYTANLAAF中的A7）与外部甲烷乙硫磺酸（MTS）试剂的反应速度在存在时比不存在激动剂NMDA和甘氨酸时快500至1000倍。事实上，MTS修饰率表现出与通道激活完全相同的NMDA浓度依赖性。相比之下，无论是否存在NMDA和甘氨酸，A651外部的残基总是以相似的动力学进行修饰。有趣的是，A651C（NR2B）的MTS修饰使通道本构打开。A651（NR2B）突变为精氨酸、色氨酸或苯丙氨酸，NR1中相应的A652的类似突变也会导致组成性通道开放。双突变周期分析进一步表明，如果A652（NR1）和A651（NR2B）突变成具有大侧链或补偿电荷的残基，则其影响明显是非加性的（即合作性的）[e]。g, A652E (NR1) + A651R (NR2B)]。因此，A7侧链在这个水平上对亚基间距离起决定作用，它直接负责NMDA受体的激活门和激活-失活门。
The NMDA receptor opens in response to binding of NMDA and glycine. However, it remains unclear where and how gating of the NMDA receptor pore is accomplished. We show that different point mutations between S645 and I655 (thus including the highly conserved SYTANLAAF motif) of M3c in NR2B lead to constitutively open channels. The current through these constitutively open channels are readily blocked by external Mg2+ and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]. Also, the open-channel blocker MK-801 can no longer be trapped in these channels when NMDA and glycine are washed off. Moreover, M3c residues at or below A651(NR2B, A7 in SYTANLAAF) react with external methanethiosulfonate (MTS) reagents similar to 500 to 1000-fold faster in the presence than in the absence of agonists NMDA and glycine. In fact, the MTS modification rate shows exactly the same NMDA concentration dependence as channel activation. In contrast, those residues external to A651 are always modified with similar kinetics whether NMDA and glycine are present or not. Interestingly, MTS modification of A651C(NR2B) holds the channel constitutively open. Mutations of A651(NR2B) into arginine, tryptophan, or phenylalanine, and similar mutations of the corresponding A652 in NR1 also lead to constitutively open channels. Double-mutant cycle analysis further shows that the effects of A652(NR1) and A651(NR2B) mutations are evidently non-additive (i.e., cooperative) if mutated into residues with large side chains or with compensatory charges [e. g., A652E(NR1)+A651R(NR2B)]. The side chain of A7 thus plays a determinant role in the intersubunit distance at this level, which is directly responsible for the activation gate and activation-deactivation gating of the NMDA receptor.