The GluR2cryst homotetramer in complex with the competitive antagonist ZK200775 was crystallized and its structure was determined at 3.6-Å resolution [75
]. The structure represents the closed state of the channel. The global architecture can be subdivided into three layers: the NTD layer, LBD layer, and the transmembrane (or channel pore) layer (Fig. ). The NTD layer and the LBD layer are each formed by a pair of NTD dimers and a pair of LBD dimers, respectively. The protomers within the NTD and LBD dimers are related to each other by the 2-fold axis perpendicular to the membrane plane. Using the same symmetry axis, the TMD has approximately 4-fold rotational symmetry, whereas the architecture of the top part of the channel pore with the linker sequence that connects to the LBD has a 2-fold rotational symmetry. The relative arrangement of the two NTD dimers in the GluA2cryst was very similar to what was observed in the crystal structure of the tetrameric GluA2-NTD [71
]. The interface between the two NTD dimers was made by the inner two subunits that are closer to the global 2-fold axis. The structures of each NTD dimer were indistinguishable from what was observed in the dimeric crystal structures of the NTD. The C-termini of the NTDs connect to the N-termini of the LBDs through the linker sequence. In this connection, the pair of subunits that form single NTD dimer is not the pair of subunits that form the LBD dimers. The structures of the LBD dimers were very similar to the previously reported structure except that the antagonist ZK200775 locked the pocket of the clamshell-like structure of the LBD into a further extended conformation than the other competitive antagonists. A novel small interface between the two LBD dimers was identified and was predicted to form a weak dimer–dimer interaction.
The domain arrangement in wild-type GluA2 was proposed by extending the models from the GluA2cryst structure. Cysteine residues were introduced into the wild-type GluA2 sequence at locations that were predicted to make close inter-domain contacts, specifically the inter-NTD dimer contact point (V209C), inter-LBD dimer contact point (K663C and I664C), and the inter-TMD contact point (M629C). When expressed in HEK cells, the mutant subunits formed disulfide bonds, indicating that the residues mutated to cysteines were within close proximity.
The architecture of the transmembrane domain of the GluR2cryst contained many of the concepts gained from the previous studies that proposed the topology of the transmembrane segments [26
]. Specifically, the M1, 3, and 4 form alpha-helices and span the membrane and the M2 is part of a reentrant loop. The structure around M2 that corresponds to the ion selectivity filter in the potassium channel [119
] was largely disordered in the crystal structure of GluA2cryst and its structure remained unclear. The channel pore was lined by the M3 helices. The alpha-helices of M3 cross near the outer side of the membrane and form a narrow constraint creating an occlusion of the putative ion permeation pathway. The region around the crossing of the M3 helices is made of the highly conserved amino acid sequence among the glutamate receptors (SYTANLAAF) that is mutated in the lurcher
mutant mice [120
]. The pore diameter of the ion permeation pathway was the narrowest where the conserved amino acids SYTANLAAF were located in the GluA2cryst structure. M4 is not part of the central ion permeable pore but an extensive interaction between the M4 helix and the others were detected.
The core gating machinery that has 2-fold symmetry is made of the region between the LBD and M3. The connection between the LBD and the M3 contributes largely to the transition of symmetry between the LBD layer (2-fold rotational symmetry) and the transmembrane layer (4-fold rotational symmetry). The lengths of the M3 alpha-helices are not equal in all four subunits. According to their length, the four M3s can be subdivided into two pairs. The pairs are defined such that within each pair the two M3s have a 2-fold rotational symmetry around the global rotational axis perpendicular to the membrane plane. In other words, if one draws a square by connecting the four M3s that appear in a cross section tangent to the membrane plane, the two M3s that are located diagonally form one pair. The length of the M3 in one of the pairs is longer than the other. The shorter M3s are connected to the two LBDs that do not form the inter-LBD dimer interface. Because the ends of these two LBDs that connect to the shorter M3s are located farther away from the channel pore, these M3–LBD linkers are more extended compared to the other two M3–LBD linkers that connect the LBDs with the longer M3s. Consequently, the two LBDs that form the interface between the two LBD dimers (Fig. , top middle structure, rectangle) are derived from the two subunits that adopt the longer M3 helices and the M3–LBD connection in these subunits are shorter. In relation to the NTDs, the subunits that adopt the shorter M3 are the same subunits as those that contribute to forming the interface between the two NTD dimers (Fig. , top right structure, rectangle). When the polypeptides of an individual subunit are traced carefully, it is immediately recognized that each subunit contributes to the overall tetrameric structure in either one of the two modes (in Fig. , subunits A and C are one mode, whereas B and D are another).
Insights into the mechanism of gating were gained from the architecture of the channel core and the geometrical arrangements of the linkers connecting the membrane spanning segment and the LBD. The displacement of the M3 helices that form the narrow constriction of the ion permeable path will be necessary for gating. The crystal structure of the GluA2cryst predicts that the shorter M3 alpha-helices will undergo larger displacement upon the glutamate-induced closure of the clamshell-like LBDs. Conversely, the longer M3 alpha-helices will have a smaller degree of displacement and thus may contribute less to the gating. In this model, the pair of LBDs that are connected to the longer and shorter M3s, respectively, contributes differently to the gating. Based on the global domain arrangements of the LBD and the cysteine crosslinking experiments, it was suggested that in the NMDA-Rs the two GluN1-derived LBDs form the interface between the two LBD dimers and thus connect to the longer M3 alpha-helices. GluN1 is the glycine binding subunit of NMDA-Rs. Because the GluA2cryst structure predicts that the LBDs connecting to the longer M3 contribute less to gating, a similar principal may explain why glycine contributes less to channel gating in the absence of glutamate.
Insights into the mechanism of desensitization were gained by comparing the crystal structure of GluAcryst and mutant LBD carrying the S729C mutation, a mutant that locks the LBD in a conformation that mimics the desensitized state [52
]. The LBD clamshell is made of two lobes denoted as D1 and D2 lobes [25
]. The D2 lobe is the lower lobe of the LBD clamshell that is closer to the membrane. The D2 lobes of the GluA2cryst in complex with the competitive antagonist ZK200775 superimpose well with the D2 lobes of LBD S729C mutant in complex with glutamate. The clamshell of the LBD S729C is closed by glutamate but the D2 lobes are separated in the same way as the LBDs in the GluA2cryst whose clamshell is open and ion channel is closed. This observation and the results from previous studies [52
] collectively suggest that desensitization results from the rupture of the LBD dimer that is made by the D1 lobes. Importantly, the GluA2cryst structure predicts that when the rupture of the D1 lobes happens during desensitization, the NTD together with the NTD–LBD linker must move. More precisely, during desensitization, it was predicted that the distances between and within the NTD dimers must change. This prediction is also supported by the previous single particle EM study that experimentally demonstrated the distinct conformation of the NTD dimers in the presence or absence of glutamate and CTZ [66
], and the electrophysiological study that demonstrates the importance during gating of multimerization state of the NTDs [121
]. It is well known that the binding of Zn2+
and ifenprodil to the NTDs can modulate channel function of the NMDA-Rs [122
]. By inverting the cause and consequence, the predicted mandatory movement of the NTDs upon desensitization is also consistent with the channel-modulating function of the NTDs in NMDA-Rs.
The crystal structure of GluA2cryst raises many new questions. The current structure of GluA2cryst represents a channel-blocked structure. Different conductive states have been reported for AMPA-Rs, suggesting the existence of more than one conformation when the channel is open [127
]. Understanding the architecture of the different gating states will be the next challenge. As described in the earlier sections, gating of AMPA-Rs is modulated by auxiliary subunits. Which part of the AMPA-Rs is involved in the functional modulation by the auxiliary subunits such as stargazin/TARPs? The cytoplasmic C-terminus of the stargazin/TARPs is critical for modulating AMPA-R gating [99
], and thus any potions of the AMPA-R that are exposed to the cytoplasm are candidate interacting targets of stargazin/TARPs. In the GluA2cryst structure, however, the cytoplasmic C-terminal and part of the M2 reentrant loop are unresolved. Functional interactions exist between stargazin/TARPs and the residues in the narrow constriction of the AMPA-Rs [129
]. The Q/R editing site located near the M2 is critical for the polyamine block of the AMPA-Rs [130
]. The polyamine block of AMPA-Rs is also modulated by stargazin/TARPs [131
]. Collectively, the opening of the pore on the cytoplasmic side is the likely candidate for the cytoplasmic interaction between stargazin and AMPA-Rs. The extracellular loops of stargazin/TARPs also participate in channel modulation [132
]. The structural and biochemical data that support the complex mechanisms of modulation remain to be seen.
The structure of the GluA2cryst provided mechanistic interpretation to various experimental observations made on the structure–function relationship of the glutamate receptors. Many principles that govern the assembly and architecture of the GluR2cryst are likely extendable to other glutamate receptors. However, the structure and mechanism of the NTDs of the NMDA-Rs are suggested to be different from AMPA-Rs [133
]. The NMDA-Rs are obligate heterotetramers that require the essential subunit GluN1. The crystal structure of the heterodimer formed of the LBDs of GluN1 and GluN2A together with the cysteine crosslinking study (E699C) of the GluN1 subunit suggests that, at the LBD level, the NMDA-Rs are assembled as a dimer of heterodimeric LBDs [59
]. In contrast, currently there is no structural evidence that supports that the arrangement of the NTDs in the NMDA-Rs follows the same principle as the AMPA-Rs. However, electrophysiological recordings from GluN2 subunits carrying mutation in the NTD suggest a model in which NTDs of GluN1 and GluN2 heterodimerize [134
]. The affinity of the NTD dimer in kainate receptors is much lower than that of the AMPA-Rs [74
]. If the affinity between the NTD dimer is lower in non-AMPA-type glutamate receptors, it is possible that depending on the conformational state and the phase of the assembly process the domain arrangement of the NTDs will dynamically change. In this view, the proposed domain arrangements in the NMDA-Rs may represent one state of the receptor and the mechanism around the NTDs will require further experimental characterization.