Riou M et al. (2012), An alternating GluN1-2-1-2 subunit arrangement ...

XB-ART-46125

PLoS One 2012 Jan 01;74:e35134. doi: 10.1371/journal.pone.0035134.

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An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors.

Riou M , Stroebel D , Edwardson JM , Paoletti P .

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NMDA receptors (NMDARs) form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular) and functional (plasma-membrane inserted) pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors.

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Species referenced: Xenopus laevis
Genes referenced: gnl3 ttn

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	Figure 1. Subunit organization of the tetrameric GluA2 AMPA receptor.Side view of the gating core (ABD + TMD) of the GluA2 homotetramer [4]. The subunits adopt two different conformations, A/C (blue) and B/D (red). The insets on the right show how subunits are organized in the ABD and TMD (pore) layer. The grey shading indicates the two local ABD dimers. The black dot indicates the point of contact between subunits A and C at the dimer-of-dimers interface in the ABD layer. Numbers 1 and 2 highlight the two regions where mutations have been introduced in NMDARs.
	Figure 2. Insertion of a single cysteine in the GluN1 M3-S2 linker results in the creation of a high-affinity copper binding site.(A) The ion-channel (M3 and M3-S2 linkers) region of a modelled tetrameric GluN1/GluN2A receptor viewed from the side (left) and from above the membrane plane (right). In this alternating model (GluN1/2/1/2), the two GluN1 subunits (blue) are in the âproximalâ A/C conformation and the two GluN2A subunits (red) in the âdistalâ B/D conformation. Homologous mutations GluN1-P660C and GluN2A-F658C are highlighted (sulphur in yellow). (B and C) Effects of reducing (B) and oxidizing (C) treatments on current amplitudes of the various cysteine (filled bars) or alanine (empty bars) mutant receptors. Values in B are (from left to right): 1.08 Â± 0.05 (nâ=â8), 0.96 Â± 0.15 (nâ=â7), 1.35 Â± 0.2 (nâ=â14), 1.38 Â± 0.16 (nâ=â8), 1.05 Â± 0.22 (nâ=â9), 1.05 Â± 0.06 (nâ=â11), 1.35 Â± 0.16 (nâ=â20), 1.49 Â± 0.2 (nâ=â6). Values in C are (from left to right): 1.18 Â± 0.18 (nâ=â32), 1.09 Â± 0.08 (nâ=â8), 1.1 Â± 0.11 (nâ=â13), 1.13 Â± 0.08 (nâ=â4), 0.51 Â± 0.11 (nâ=â28), 0.90 Â± 0.15 (nâ=â19), 0.65 Â± 0.15 (nâ=â20) and 0.56 Â± 0.08 (nâ=â8). (D) Copper inhibition dose-response curves for wild-type (open circles; Inhibmax â=â 21%, IC50 â=â 0.19 nM, nH â=â 1.3), GluN1wt/GluN2A-F658C (open squares; Inhibmax â=â 34%, IC50 â=â 0.33 nM, nH â=â 0.7), GluN1-P660C/GluN2Awt (filled circles; Inhibmax â=â 78%, IC50 â=â 0.075 nM, nH â=â 3) and GluN1-P660A/GluN2Awt receptors (filled squares; Inhibmax â=â 15%, IC50 â=â 0.21 nM, nH â=â 1.5). Right: current traces illustrating the inhibition of GluN1-P660A/GluN2Awt (top) and GluN1-P660C/GluN2Awt (bottom) receptors by 0.3 nM free copper. *** corresponds to P<0.001; ns, non-significant; Studentâs t-test.
	Figure 3. Disulfide cross-linking at the putative ABD dimer-of-dimers interface between GluN1, but not GluN2A, subunits.(A) Three possible arrangements viewed from the membrane region of GluN1/GluN2A ABD heterodimers in a NMDAR based on GluA2 structure. Mutated residues GluN1-E698 and GluN2A-Q671 are highlighted in blue and red, respectively. The residues either lie at the dimer-of-dimers interface in the proximal A/C subunits or protrude towards the exterior surface of the receptor in the distal B/D subunits. (B) MK-801 inhibition kinetics for various GluN1 (top) and GluN2A (bottom) mutants. Values are (from left to right): 0.9 Â± 0.04 (nâ=â5), 0.93 Â± 0.09 (nâ=â9), 1.85 Â± 0.13 (nâ=â20), 0.91 Â± 0.05 (nâ=â8), and 0.87 Â± 0.06 (nâ=â7) for GluN1 mutants; 1.17 Â± 0.15 (nâ=â21), 0.90 Â± 0.06 (nâ=â11), 0.91 Â± 0.09 (nâ=â11) and 0.87 Â± 0.1 (nâ=â11) for GluN2A mutants. All GluN1 mutant subunits were co-injected with GluN2A wt and all GluN2A mutant subunits with GluN1 wt. (C) Effects of redox treatments on the MK-801 inhibition kinetics for cysteine (filled bars) or alanine (empty bars) mutants. Values are (from left to right): control conditions, 1.85 Â± 0.13 (nâ=â 20), 0.96 Â± 0.07 (nâ=â 12), 1.17 Â± 0.15 (nâ=â 21), 1.19 Â± 0.06 (nâ=â 9); after incubation with Cu/Phen (oxidation): 2.21 Â± 0.18 (nâ=â 12), 1.07 Â± 0.07 (nâ=â 10), 1.28 Â± 0.14 (nâ=â 18), 1.36 Â± 0.14 (nâ=â 9); after subsequent incubation with TCEP (reduction): 1.31 Â± 0.18 (nâ=â 19), 0.88 Â± 0.08 (nâ=â 7), 0.83 Â± 0.09 (nâ=â 17), 0.86 Â± 0.11 (nâ=â 9). Inset: MK-801 inhibition kinetics are slower in non-treated GluN1-E698C/GluN2Awt receptors (2) than in non-treated GluN1-E698A/GluN2Awt receptors (1) or TCEP-treated GluN1-E698C/GluN2Awt receptors (3) (normalized currents). (D) Effects of MTSET application on current amplitudes for cysteine (filled bars) or alanine (empty bars) mutants. Values are (from left to right): 0.70 Â± 0.02 (nâ=â18), 0.95 Â± 0.03 (nâ=â11), 0.95 Â± 0.04 (nâ=â9) and 0.95 Â± 0.02 (nâ=â5). Right: current traces illustrating the effects of MTSET on GluN1-E698A/GluN2Awt and GluN1-E698C/GluN2Awt receptors. *** corresponds to P<0.001; ns, non-significant; Studentâs t-test.
	Figure 4. Intracellular and plasma-membrane pools of disulfide cross-linked receptors.(A) Immunoblots from Xenopus oocytes expressing either wt or GluN2A mutant receptors. (B) Schematic representation of a cell expressing intracellular and plasma-membrane pools of receptors with intersubunit disulfide cross-links. Because of their differential membrane permeability, the reducing agent DTE acts on both pools while the reducing agent TCEP acts solely on the cell surface pool. (C) Immunoblots from oocytes expressing either wt or GluN1 mutant receptors. (D) Immunoblots from occytes injected either with both GluN1 and GluN2A subunits or with the GluN1 subunit alone. M1 indicates the expected band position of the GluN1 monomer (â¼110 kDa); M2, the GluN2A monomer (â¼180 kDa); D1, the GluN1 homodimer (â¼220 kDa) and T1 the approximate location of the GluN1 homo-trimer and/or homo-tetramer (>300 kDa). CC stands for mutations GluN1-N521C-L777C (in ABD intradimer interface). M stands for molecular weight marker and n.i. for non-injected oocytes. * indicates non-specific bands seen with the anti-GluN1 antibody.

References [+] :

Banke, Activation of NR1/NR2B NMDA receptors. 2003, Pubmed