NMDA離子通道之門閥調控機轉

NMDA離子通道分別是由兩個GluN1和兩個GluN2所組成，然而這兩個不同的次單元在通道上的貢獻還不是很清楚。我們發現glycine對GluN1的親和力(K gly ∼ 0.6 μM)明顯高於NMDA或glutamate對GluN2的親和力(K NMDA ∼ 36 μM, K glu ∼ 4.8 μM)。然而Glycine對GluN1的結合速度(∼9.8 × 106 M-1 s-1) 稍為快於NMDA 對 GluN2 (∼4.1 × 106 M-1 s-1)。Glycine從GluN1離開結合位的速度(time constant ∼2 s)明顯慢於NMDA從GluN2離開結合位的速度(time constant ∼70 ms)。除此之外，同時洗掉glycine和NMDA的NMDA通道decay速度和NMDA離開結合位的速度相似。但是如果只洗掉glycine，NMDA通道decay速度明顯變慢，且和glycine離開結合位相似。在缺乏給予NMDA的情況下，glycine的結合速度對於離開結合位的通道，大概和共同給予NMDA和glycine相似。然而在缺乏給予glycine下的NMDA結合速度明顯變慢。另外，NMDA離子通道的GluN1和GluN2上有一段保留序列SYTANLAAF，其第七個胺基酸A7在GluN1和GluN2上非常靠近可以控制外口的大小。除此之外，突變在GluN1的A7可以改變glycine和NMDA對離子通道的親和力，但在GluN2則只有影響NMDA對通道的親和力。因此，GluN2是負責活化的門閥，而GluN1扮演調控門閥的角色。結構上，調控次單元似乎是透過A7的胺基酸。除此之外，NMDA對於鈣離子有相當高的通透性，而鈣離子的通透會影響許多細胞生物的性質。NMDA具有鈣離子依賴性的去敏感性，而目前研究顯示鈣離子和胞內的calcineurin等物質會交互作用產生鈣離子依賴性的去敏感性，然而由於在外口也有鈣離子的結合位，鈣離子是否也會在外口作用，產生鈣離子依賴性的去敏感性。我們發現在外口的鈣離子和鉻離子（和鈣離子有相同價電子數和半徑）會結合到休息態的NMDA而影響門閥的開關。由於活化門閥位於外口處，這些離子的結合位應該處於更外口的地方。根據我們結果顯示鉻離子鉻離子對通道是一對一的結合，其對休息態,活化態,去敏感態的親和力分別為分別為~5, ~2.5, and ~1.2 μM。DRPEER motif(在GluN1而不在GluN2)位於活化門閥的外面，當DEE突變成Alanine時則Cd離子的親和力無論是休息態或是活化態會下降，因此鈣或鉻離子可能透過DRPEER motif進而影響活化門閥，鈣或鉻離子的抑制能力在T647A突變也會下降，且會產生明顯的hook電流，暗示這突變的通道只有休息態和活化態，沒有去敏感態，也暗示去敏感態和conformational的改變有關。另一方面，去敏感態在NMDA扮演重要的角色，目前切確的調控機轉仍然未知，我們發現tetrapentylammonium (TPentA)接在外口會有gating modify的角色，使通道喜歡待在開啟的裝態，而不喜歡待在去敏感態。另外SYTANLAAF在gating上也扮演很重要的角色，像是A7或是T3突變之後對desensitization都有嚴重的影響，暗示desensitization是一種結構上的改變，而且可能就在活化閘門A7的附近。

關鍵字

離子通道

並列摘要

The NMDA receptor channel is an obligatory heterotetramer formed by two GluN1 and two GluN2 subunits. However, the differential contribution of the two different subunits to channel operation is not clear. We found that the apparent affinity of glycine to GluN1 (Kgly~0.6 μM) is much higher than NMDA or glutamate to GluN2 (KNMDA~36 μM, Kglu~4.8 μM). The binding rate constant (derived from the linear regression of the apparent macroscopic binding rates) of glycine to GluN1 (~9.8 x 106 M-1s-1), however, is only slightly faster than NMDA to GluN2 (~4.1 x 106 M-1s-1). Accordingly, the apparent unbinding rates of glycine from activated GluN1 (time constant ~2s) are much slower than NMDA from activated GluN2 (time constant ~70 ms). Moreover, the decay of NMDA currents upon wash-off of both glycine and NMDA seems to follow the course of NMDA rather than glycine unbinding. But if only glycine is washed off, the current decay is much slower, apparently following the course of glycine unbinding. The apparent binding rate of glycine to the fully deactivated channel, in the absence of NMDA, is roughly the same as that measured with co-application of both ligands, whereas the apparent binding rate of NMDA to the fully deactivated channel in the absence of glycine is markedly slower. In this regard, it is interesting that the 7th residue in the highly conserved SYTANLAAF motif (A7) in GluN1 and GluN2 are so close that they may interact with each other to control the dimension of the external pore mouth. Moreover, specific mutations involving A7 in GluN1 but not in GluN2 result in channels showing markedly enhanced affinity to both glycine and NMDA and readily activated by only NMDA, as if the channel is already partially activated. We conclude that GluN2 is most likely directly responsible for the activation gate of the NMDA channel, whereas GluN1 assumes a role of more global control, especially on the gating conformational changes in GluN2. Structurally, this inter-subunit regulatory interaction seems to involve the SYTANLAAF motif, especially the A7 residue. Furthermore, the NMDA receptor channel is characterized by its high permeability of Ca2+ ion, and the Ca2+ influxes may play an important role in many cellular physiological and pathophysiological processes. It has been reported that NMDA channel desensitization, an imperative attribute regulating ionic fluxes through the channel pore, could be related to Ca2+ ions flowing through the pore and/or interacting with effector proteins such as calcineurin at the internal pore mouth. Whether extracellular Ca2+ ion itself could directly bind to the NMDA receptor channel to have an effect on the key molecular behaviors of the channel has not been fully characterized. We found that extracellular Ca2+ and Cd2+, an ion with the same charges and ionic radius as Ca2+ and therefore high affinity toward many Ca2+ binding sites, could bind to the closed NMDA channel to affect channel gating as well as ion permeation. Because the activation gate, which is already positioned at the external pore mouth, is not open, this binding site must be located at the very external part of the pore. We also demonstrated that Cd2+ binds to the NMDA channel with a simple bimolecular reaction, and the apparent dissociation constants toward the closed, open, and desensitized states of the channel is ~5, ~2.5, and ~1.2 μM, respectively. The modest but definite differential affinity would indicate that the binding site changes its conformation during the gating process. DRPEER, a motif in the GluN1 but not GluN2 subunit, is located just external to the activation gate of the NMDA channel. Interestingly, the effect of Cd2+ present in either the resting or the activated state is decreased correlatively to the number of charge-neutralization mutations in this motif, with additive free energy changes calculated by double mutant cycle analysis. DRPEER motif therefore very likely constitutes the binding site for Ca2+ or Cd2+, and thus seems to go through a sequential conformational change during channel activation. In this regard, it is intriguing that the inhibitory effect of Cd2+ is also decreased by point mutation T647A located just inside the activation gate in the pore. Moreover, prominent “hooked” current develops after wash-off of Cd2+ (and even more so after wash-off of Ca2+) in this case, suggesting preservation of the channel in the open state (prevention of the channel from entering the desensitized state) and thus an apparently inversed order of affinity of Cd2+ and Ca2+ toward the two states with the point mutation. Desensitization thus seems to involve essential conformational changes in the vicinity of the activation gate in the NMDA channel. Desensitization is an important gating mechanism for the function of N-methyl-D-aspartate (NMDA) receptors. However, the exact modulation of desensitization mechanism remains in question. We found that extracellular tetrapentylammonium (TPentA) binds to the external pore mouth, and induces a strong increase in open probability to disturb the equivalence between open and desensitization states. TpentA seems to prefer to open state of the NMDA channel. Moreover, the A7 residues (at position 7 of the SYTANLAAF motif), shown to mark the activation gate, constitute the key component of the activation gate of the NMDA channel. We further explore whether A7 is also involved in the desensitization. Our data suggest that the desensitization gate should be near the activation gate, A7, in the external pore mouth.

並列關鍵字

NMDA receptor

參考文獻

1. Anson LC, Chen PE, Wyllie DJ, Colquhoun D, Schoepfer R (1998) Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors. J Neurosci 18 (2):581-589

2. Armstrong CM (1971) Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. The Journal of general physiology 58 (4):413-437

3. Ascher P (1990) Measuring and controlling the extracellular glycine concentration at the NMDA receptor level. Advances in experimental medicine and biology 268:13-16

4. Beck C, Wollmuth LP, Seeburg PH, Sakmann B, Kuner T (1999) NMDAR channel segments forming the extracellular vestibule inferred from the accessibility of substituted cysteines. Neuron 22 (3):559-570

5. Benveniste M, Clements J, Vyklicky L, Jr., Mayer ML (1990) A kinetic analysis of the modulation of N-methyl-D-aspartic acid receptors by glycine in mouse cultured hippocampal neurones. The Journal of physiology 428:333-357

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NMDA離子通道之門閥調控機轉

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