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The differential contribution of GluN1 and GluN2 to the gating operation of the NMDA receptor channel

  • Ion channels, receptors and transporters
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Abstract

The Ν-methyl-d-aspartate (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 (K gly ∼ 0.6 μM) is much higher than NMDA or glutamate to GluN2 (K NMDA ∼ 36 μM, K glu ∼ 4.8 μM). The binding rate constant (derived from the linear regression of the apparent macroscopic binding rates) of glycine to GluN1 (∼9.8 × 106 M−1 s−1), however, is only slightly faster than NMDA to GluN2 (∼4.1 × 106 M−1 s−1). Accordingly, the apparent unbinding rates of glycine from activated GluN1 (time constant ∼2 s) 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 seventh 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 intersubunit regulatory interaction seems to involve the SYTANLAAF motif, especially the A7 residue.

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Abbreviations

AMPA:

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

DCKA:

5,7-Dichlorokynurenic acid

DMSO:

Dimethyl sulfoxide

MTS:

Methanethiosulfonate

MTSEA:

MTS-ethylammonium

MTSET:

MTS-ethyltrimethylammonium

NMDA:

Ν-methyl-d-aspartate

TAA:

Tetraalkylammonium

TPA:

Tetrapropylammonium

TBA:

Tetrabutylammonium

TpentA:

Tetrapentylammonium

ThexylA:

Tetrahexylammonium

TheptylA:

Tetraheptylammonium

TOA:

Tetraoctylammonium

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Acknowledgments

This work was supported by Grants NHRI-EX102-10006NI and NHRI-EX103-10006NI from the National Health Research Institutes and Grant NSC100-2320-B-002-009-MY3 from the National Science Council, Taiwan.

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Authors and Affiliations

  1. Department of Physiology, National Taiwan University College of Medicine, 1, Jen-Ai Road, 1st Section, Taipei, 100, Taiwan

    Ya-Chi Tu & Chung-Chin Kuo

  2. Department of Neurology, National Taiwan University Hospital, Taipei, 100, Taiwan

    Chung-Chin Kuo

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  1. Ya-Chi Tu
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Correspondence to Chung-Chin Kuo.

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Fig. S1

Generation of constitutively open channels by MTS modification of A652C (GluN1) and A651C (GluN2) mutations. a Before MTSET modification, the same experiment as that described in Fig. 8a was repeated in GluN1(A652C), and GluN1(A652C)/ GluN2(A651E) mutant channels. The thick line, gray bar, white bar, and black bar represent application of Mg2+-free NMG-Tyrode, Mg2+-free Na+-Tyrode, Mg2+-free Na+-Tyrode solution containing 300 μM NMDA + 30 μM glycine, and Na+-Tyrode solution containing 2 mM Mg2+, respectively. b MTSET reagents were applied with agonists (300 μM NMDA + 30 μM glycine) for 12 sec. The white and red bars above the current trace indicate application of the agonists and MTSET, respectively. MTSET has a rapid and very small inhibitory effect on the WT channel. The effect, however, is completely reversible and thus most likely ascribable to mild pore block rather than protein modification by the MTS agents. On the other hand, MTS modification irreversibly enhances the currents in the GluN1(A652C) single mutant channel with a time constant of ∼6.2 sec. In sharp contrast, MTS modification irreversibly inhibits (rather than enhances) the currents in the GluN1(A652C)/ GluN2(A651E) double mutant channel with a much shorter time constant of 754 ms. c After MTSET modification, the same experiment as that described in Fig. 8a was repeated in GluN1(A652C), and GluN1(A652C)/ GluN2(A651E) mutant channels. The thick line, gray bar, white bar, and black bar represent application of Mg2+-free NMG-Tyrode, Mg2+-free Na+-Tyrode, Mg2+-free Na+-Tyrode solution containing 300 μM NMDA + 30 μM glycine, and Na+-Tyrode solution containing 2 mM Mg2+, respectively. d Cumulative results are obtained with the same experimental protocol described in part b (n = 3-6 for each different mutants). Note the modification rates are much higher in the GluN1(A652C)/ GluN2(A651E) and GluN1(A652E)/ GluN2(A651C) double mutant channels than in the GluN1(A652C) or GluN2(A651C) single mutant channels. e Cumulative results are obtained with the same experimental protocol described in part c (n = 3-6 for each different mutants). f Double-mutant cycle analysis of different mutant channels after MTS modification. (PDF 283 kb)

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Tu, YC., Kuo, CC. The differential contribution of GluN1 and GluN2 to the gating operation of the NMDA receptor channel. Pflugers Arch - Eur J Physiol 467, 1899–1917 (2015). https://doi.org/10.1007/s00424-014-1630-z

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