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Structure of an agonist-bound ionotropic glutamate receptor

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Science  29 Aug 2014:
Vol. 345, Issue 6200, pp. 1070-1074
DOI: 10.1126/science.1256508

Activating a receptor to excite a neuron

Transmitting signals between nerve cells, occuring at structures known as synapses, is critical to processes such as learning and memory. Fast transmission occurs when glutamate is released from a presynaptic neuron and binds to ionotropic glutamate receptors (iGluRs) in the cell membrane of a postsynaptic neuron. The iGluR contains an ion channel that is transiently opened, to activate the postsynaptic neuron, but then closes rapidly. Chen et al. and Yelshanskaya et al. report crystal structures in a range of conformations that together provide insight into how glutamate binding causes the channel to open and how other molecules that bind to the receptor modulate this. The information could aid in the design of drugs to treat cognitive impairment or seizure disorders

Science, this issue p. 1021 and p. 1070

Abstract

Ionotropic glutamate receptors (iGluRs) mediate most excitatory neurotransmission in the central nervous system and function by opening their ion channel in response to binding of agonist glutamate. Here, we report a structure of a homotetrameric rat GluA2 receptor in complex with partial agonist (S)-5-nitrowillardiine. Comparison of this structure with the closed-state structure in complex with competitive antagonist ZK 200775 suggests conformational changes that occur during iGluR gating. Guided by the structures, we engineered disulfide cross-links to probe domain interactions that are important for iGluR gating events. The combination of structural information, kinetic modeling, and biochemical and electrophysiological experiments provides insight into the mechanism of iGluR gating.

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate most excitatory neurotransmission in the central nervous system (1). iGluRs are implicated in nearly all aspects of nervous system development and function, and their dysfunction is associated with devastating chronic neurodegenerative conditions, such as Alzheimer’s and Parkinson’s diseases; psychiatric disorders, such as schizophrenia and depression; and acute disorders, such as brain trauma and stroke (13). The iGluR family includes three major subtypes: AMPA, kainate (KA), and N-methyl-d-aspartate (NMDA) receptors, which have diverse kinetic and pharmacological properties and play disparate roles in cognition but share common structural features (1).

iGluRs function as assemblies of four subunits. Whereas the majority of iGluRs in the nervous system are heterotetramers composed of at least two types of subunits, AMPA and select KA subunits can form functional homotetramers. Each iGluR subunit has a modular design (4) and includes an extracellular amino-terminal domain (ATD) that controls subunit assembly, receptor trafficking, channel gating, agonist potency, and allosteric modulation; a clamshell-shaped ligand-binding domain (LBD or S1S2) composed of two stretches of polypeptide, S1 and S2, and responsible for agonist and antagonist recognition; a transmembrane domain (TMD) that forms a cation-conducting channel and contributes to iGluR tetrameric stability; and a cytoplasmic C-terminal domain involved in synaptic localization, trafficking, mobility, and receptor regulation (1). Currently, structural information on iGluRs is limited to high-resolution crystal structures of genetically excised, water-soluble ATDs and LBDs and 3.6 to 4.0 Å resolution structures of an intact AMPA receptor in the antagonist-bound closed state (5) and NMDA receptors in the allosterically inhibited agonist-bound closed state (6, 7).

iGluRs function by opening their ion channel in response to binding of agonist glutamate (Glu) (1). This process of activation gating is accompanied by the typically slower process of desensitization, which leads to closure of the ion channel and causes a reduction of iGluR-mediated currents in the continuous presence of Glu. Desensitization represents a versatile mechanism for shaping synaptic transmission (8), and controlling it could be an effective way of regulating synaptic integration, modulating circuit function, and altering iGluR activity in pathological conditions (1, 9). Here, we used a combination of biochemical and biophysical approaches to study iGluR gating. We focused on AMPA subtype rat GluA2 receptors, for which a modified construct, GluA2cryst, had previously yielded the structure in the closed antagonist-bound state (5). We used a construct, GluA2*, that is similar to GluA2cryst but has several mutations reversed back toward the wild-type GluA2 background (see supplementary materials).

For functional characterization, we expressed GluA2* in human embryonic kidney (HEK) 293 cells and used patch clamp with fast solution exchange to record GluA2*-mediated currents. As is typical for AMPA subtype iGluRs at near-physiological membrane potentials, Glu application elicited an inward current that quickly decayed in the continuous presence of Glu as a result of desensitization (Fig. 1A). Desensitization in GluA2* receptors had a similar rate and extent as in wild-type receptors (fig. S1, A and D) and was blocked by the positive allosteric modulator cyclothiazide (CTZ) (10).

Fig. 1 Functional characterization and structure of NOW-bound GluA2*.

(A) Representative whole-cell currents recorded at –60-mV membrane potential from HEK 293 cells expressing GluA2* in response to 500-ms application of 1 mM Glu alone, application of Glu in the continuous presence of 30 μM of the positive allosteric modulator CTZ, application of 100 μM NOW alone, and application of NOW in the continuous presence of CTZ. Solid and open triangles indicate steady-state currents at the end of NOW and Glu applications, respectively. (B) Occupancies of the closed (C), open (O), and desensitized (D) states of the receptor at the time points indicated by the triangles in (A). Subscript “A” and * indicate agonist-bound and ion-conducting states, respectively. The occupancies were predicted by using kinetic modeling (fig. S2). (C) The broad (left) and narrow (right) faces of the GluA2NOW structure viewed parallel to the membrane and perpendicular to the overall twofold axis of molecular symmetry. Inner and outer sides of the membrane are indicated by parallel gray bars. Each of four subunits is in a different color.

AMPA receptors can also be activated by partial agonists, which elicit smaller maximal whole-cell currents than the full agonist Glu but have similar single-channel conductance (1113). In our studies, we used a partial agonist, (S)-5-nitrowillardiine (NOW). In crystal structures of isolated LBD, NOW demonstrated the ability to keep the LBD clamshell completely closed (13), just like full agonists. NOW elicited currents with ~four times smaller maximal amplitude than Glu and had similar potency for GluA2* and wild-type GluA2 receptors (fig. S1, B and C). The rates of desensitization and, to a greater extent, recovery from it for NOW were apparently slower than those for Glu (fig. S1, D to G). Kinetic modeling of the effects of full and partial agonists on AMPA receptors (14) (fig. S2) closely reproduced our experimental data and predicted that, in the presence of saturating concentrations of either, nearly all receptors accumulate in the agonist-bound desensitized states (DA, Fig. 1B).

We purified GluA2* and crystallized it in the presence of NOW. The best crystals belonged to the C2221 space group and diffracted to 4.8 Å resolution (table S1). We solved the structure (GluA2NOW) by molecular replacement, using the high-resolution structure of the isolated ATD dimer [Protein Data Bank (PDB) identification code (ID) 3H5V] (15) and the ion channel domain from GluA2cryst (PDB ID 3KG2) (5) as search models. The NOW-bound structure of the isolated LBD (PDB ID 3RTW) (13) did not work as a molecular replacement search model for LBD, suggesting that the LBD conformation is different in the context of GluA2NOW. We solved the structure by separately using the D1 and D2 lobes of LBD as molecular replacement search models. The conformation of LBD in the GluA2NOW structure most closely resembles the 6-cyano-7-nitroquinoxaline-2,3-dione–bound conformation of the isolated LBD (PDB ID 3B7D) (16), and their superposition yields a root mean square deviation (RMSD) of 0.31 Å. Phases were improved by multidomain noncrystallographic symmetry averaging, solvent flattening, and histogram matching. The electron density maps were of sufficient clarity to build the majority of linkers connecting the LBD to the ion channel and the ATD to the LBD (fig. S3). The resulting model was refined to good crystallographic statistics and stereochemistry (Rwork/Rfree = 0.228/0.262, table S1).

The GluA2NOW structure (Fig. 1C) is shaped like the letter Y and has a domain arrangement similar to the antagonist ZK 200775 (ZK)–bound closed-state GluA2cryst structure (5). For better comparison with GluA2NOW, we cocrystallized GluA2* with ZK (table S1). The resulting structure (GluA2ZK) closely resembles the original GluA2cryst structure (superposition of individual domains of GluA2ZK and GluA2cryst yields RMSD of 0.3 to 1 Å).

Grossly, GluA2NOW and GluA2ZK have similar architectures (Fig. 2A). The conformations of ATD are very similar (fig. S4A). The ion channel of GluA2NOW adopts a closed-pore conformation that is also similar to GluA2ZK (figs. S4, B and C, and S5). Nevertheless, small differences in the cross-pore dimensions between two pairs of diagonal subunits A/C and B/D (fig. S4, D to G) suggest deeper expansion of the twofold rotational symmetry of extracellular domains into the extracellular half of the GluA2NOW ion channel. The strongest difference in domain conformation was observed for the LBD: Each clamshell is ~11° more closed in GluA2NOW compared with GluA2ZK (Fig. 2B). The closure of individual clamshells results in wider and shorter conformations of the back-to-back LBD dimers in GluA2NOW (Fig. 2, C and D).

Fig. 2 Comparison of NOW-bound and ZK-bound GluA2*.

(A) Superposition of the full-length GluA2NOW (red) and GluA2ZK (blue). Red arrows show changes in the GluA2NOW structure compared with GluA2ZK. (B) Superposition of individual LBDs from GluA2NOW and GluA2ZK structures based on the upper lobe D1 and viewed along the axis of 11° rotation that brings the lower lobe D2 of GluA2ZK into GluA2NOW. Helices E, H, and I are labeled. (C and D) LBD dimers from the GluA2ZK (C) and GluA2NOW (D) structures. Shown are Cα’s (yellow spheres) and distances between them for E487 and I633 as well as ZK and NOW molecules as stick models.

The altered conformation of LBD dimers in GluA2NOW results in increased tension in linkers connecting LBD to ATD and TMD. Because ATD and the ion channel maintain their closed-state–like conformations, the additional tension forces pull ATD dimers down, tilting them ~1.2° away from the overall twofold axis of symmetry (Fig. 2A). The ~2.4° splaying of the ATD dimers away from each other occurs around a hinge point at the ATD dimer-dimer interface. This interface maintains nearly identical conformations in GluA2NOW and GluA2ZK, strongly supporting a critical role of ATD in iGluR tetramerization (1). Simultaneously, the tension forces pull the ion channel up by making the whole structure ~2 Å shorter and push the two LBD dimers toward each other. As a result, the interface between the two LBD dimers in GluA2NOW becomes tighter and covers a surface area three times larger than that in GluA2ZK (fig. S4, H and I). Accordingly, the GluA2NOW structure becomes ~2 Å narrower at the level of the LBD.

We tested the functional importance of agonist-induced tightening of the interface between two LBD dimers predicted by the GluA2NOW structure in experiments with GluA2-I664C (Ile664→Cys664) receptors activated by Glu. I664 residues are located in the middle of the dimer-dimer interface; the Cα’s are 13.5 Å apart in GluA2ZK and 11.3 Å apart in GluA2NOW (fig. S6A). Cross-linking of I664C has been shown in experiments with purified receptors (5). We compared the rates of GluA2-I664C receptor recovery from desensitization in reducing and nonreducing conditions. In nonreducing conditions, the recovery was incomplete (fig. S6, B to D), apparently because of slow peak current reduction. The reduction was faster with longer exposures to Glu, accompanied by a decrease in the steady-state current amplitude without substantial changes in the fast rate of desensitization, and completely reversed by dithiothreitol (DTT) (fig. S6, E and F). The majority of receptors quickly become nonconducting in the presence of agonist; thus, slow inactivation of GluA2-I664C receptors under nonreducing conditions is consistent with agonist-induced tightening of LBD dimer-dimer interface.

Compared with GluA2ZK, the LBD clamshells in GluA2NOW are ~11° more closed (Fig. 2B), presenting structural evidence of clamshell motion in the context of the intact, multidomain iGluR, consistent with studies of isolated LBDs (17). The conformational change is smaller than in agonist- or partial agonist–bound structures of isolated LBD, where clamshells are up to ~14° more closed than in GluA2NOW (fig. S7). Despite different extents of clamshell closure between the isolated LBD and GluA2NOW, the orientation of the NOW molecule in the binding pocket is similar (fig. S8). However, increased distances between atoms of NOW and the binding pocket residues (table S2) suggest weakened interactions. Perhaps the most unexpected feature of the GluA2NOW structure is the conformation of the LBD dimer interface. Although the GluA2NOW structure was crystallized at conditions apparently favoring desensitization (Fig. 1B), the D1-D1 interface appears nearly intact rather than ruptured (18). Indeed, the difference in distance between upper lobes D1 in GluA2NOW and GluA2ZK does not exceed ~1 Å, whereas the distance between lower lobes D2 is ~6 Å larger in GluA2NOW (Fig. 2, C and D).

To probe the LBD dimer–interface conformation at conditions favoring different gating states in solution, we substituted individual residues at this interface with cysteines (Fig. 3, A and B) and purified wild-type and cysteine-substituted GluA2* receptors in their tetrameric forms (fig. S9). Cysteine cross-linking was tested in the presence or absence of reducing agent (2 mM DTT) and in the presence of different ligands. The GluA2NOW and GluA2ZK structures predicted position-specific differences in cross-interface distances between substituted cysteines (Fig. 3C), but nearly all of them were capable of forming cross-links in nonreducing conditions favoring all gating states: closed, open, and desensitized (Fig. 3D). We obtained similar results for the wild-type and K493C full-length GluA2 receptors (fig. S10). Such high reactivity of cysteines at the dimer interface indicates that LBD is highly dynamic (13, 1927) and that its conformation fluctuates during gating. Because the cross-dimer disulfide bonds are unstable and reversible (20), they cannot abolish these fluctuations (Fig. 3D), but they can favor certain gating states.

Fig. 3 LBD dimer-interface cross-linking and high mobility of LBD.

(A and B) Superposition of LBD dimers from GluA2NOW (red) and GluA2ZK (blue) structures based on the upper lobes D1 and viewed perpendicular to (A) or along (B) the axes of rotation that brings the lower lobes D2 of GluA2ZK into GluA2NOW. Spheres show Cα’s for residues at the LBD interface substituted with cysteines. D, Asp; G, Gly; K, Lys; N, Asn. (C) Table showing LBD cross-interface distances between Cα’s of the cysteine-substituted residues. Each number is the average of distances for the AD and BC dimers in angstroms. (D) SDS–polyacrylamide gel electrophoresis analysis of spontaneous cross-linking of cysteines introduced at the LBD interface. The experiments were carried out with GluA2* receptors in reducing conditions (2 mM DTT, left lanes); in nonreducing conditions but in the presence of 3 mM Glu and 50 μM CTZ (Glu+CTZ, favoring the open state), 3 mM Glu (Glu, favoring the desensitized state), 500 μM NOW (NOW, favoring the desensitized state), or 100 μM ZK (ZK, favoring the antagonist-bound closed state); or in the absence of ligands (Apo, favoring the unliganded closed state). Solid and open triangles indicate positions of monomeric and dimeric bands, respectively. In the absence of reducing agent, substituted cysteines can cross-link in conditions that favor different gating states, indicating high mobility of GluA2* LBDs in solution. WT, wild type.

To study the effect of LBD interface cross-linking on iGluR gating, we recorded Glu-activated currents from HEK 293 cells expressing wild-type or cysteine-substituted receptors (Fig. 4A and fig. S11). To separate effects on receptor activation and desensitization, we measured two parameters. The first parameter gives an estimate for the effect of cross-link on AMPA receptor activation (Fig. 4B). No difference in activation between reducing and nonreducing conditions was observed for wild-type and K493C receptors. Cross-linking of cysteines in the lower D1-D1 interface (P494C and S497C; P, Pro; S, Ser) had a positive effect on activation. In a reducing environment, cysteine substitution of P494 almost entirely abolished iGluR function, whereas cross-linking recovered small currents, consistent with the very low probability of disulfide bond formation (Fig. 3D). The S497C receptors demonstrated robust Glu-activated currents that had larger amplitudes under cross-linking conditions. Overall, strengthening of the D1-D1 interface seemed to promote channel ability to open, which was most obvious when cysteine substitutions themselves inhibited this ability. In contrast, cross-linking of cysteines substituted in the D2 lobe produced negative effects on iGluR activation that were stronger for positions located closer to the ion channel. Hence, the LBD D2 lobes interlocking appears to prevent their separation, which is necessary for channel opening.

Fig. 4 Effects of LBD dimer interface cross-linking on gating.

(A) Representative whole-cell currents recorded at –60-mV membrane potential from a HEK 293 cell expressing GluA2-K493C in response to a 500-ms application of 1 mM Glu alone and applications of Glu in the continuous presence of 30 μM CTZ or 2 mM DTT or CTZ and DTT. Subscripts 0, SS, and DTT label initial, steady-state, and measured in reducing conditions current amplitudes, respectively. (B) Effects of cysteine cross-linking on activation. Shown is the ratio of initial currents recorded in the continuous presence of CTZ in the absence and presence of DTT. (C) Effects of cysteine cross-linking on GluA2 desensitization. Shown is the extent of current reduction resulting from desensitization measured in absence (solid bars) or presence (open bars) of DTT. Error bars indicate SE.

The second parameter reflects apparent changes in AMPA receptor desensitization, particularly its inhibition (Fig. 4C). No strong inhibition of desensitization was observed for wild type, K493C, or any receptors with cysteine substitutions in D2. Some of these mutant receptors had rates and extents of desensitization that were somewhat different from those of wild type (table S3), but those effects were due to mutations themselves and did not depend on the redox condition. Moreover, it is possible that the D2 cysteine cross-links can promote desensitization by locking receptors in the nonconducting state, unable to activate (Fig. 4B). Only P494C and S497C substitutions in the lower D1-D1 interface showed strongly reduced desensitization (Fig. 4C). Whereas for P494C, inhibition of desensitization was apparently independent of redox condition, it was greatly enhanced by cysteine cross-linking in S497C receptors. The inhibition of desensitization observed in P494C and S497C receptors is reminiscent of the effect of the L483Y (L, Leu; Y, Tyr) mutation (28) and of CTZ binding (10, 29), both of which take place at the D1-D1 interface.

On the basis of our mutational, structural, and functional experiments in combination with previous work, we present two possible gating models (fig. S12); both assume that LBDs are highly mobile (Fig. 3); agonist binding causes LBD clamshell closure (17); and channel opening occurs when LBD clamshells adopt their maximally closed conformation (22, 23, 3032), represented by agonist-bound structures of isolated LBD (13, 17) with the D1-D1 interface intact (fig. S7). The first model represents a traditional view (18) where the final desensitized state has the D1-D1 interface modified (fig. S12A). In this model, the GluA2NOW structure represents the agonist-bound closed state, which is predicted to be a transient state with negligible occupancy (Fig. 1B and fig. S2F) insufficient to produce protein crystals. Nevertheless, such a scenario is plausible if only a limited range of conformations of the protein is accessible in the solubilized receptor or the crystal lattice contacts substantially affect protein conformation.

The second model (fig. S12B) assumes two-step desensitization with GluA2NOW representing a deep desensitized state. This model is consistent with the predictions of kinetic modeling that, at high NOW concentrations, the majority of receptors accumulate in the deep desensitized state (D24 in fig. S2). It also predicts that the same tension forces, applied from ATD and the ion channel through the connecting linkers that open LBD clamshells during deactivation, help transition the receptor from the deep desensitized state back to the desensitized state. Therefore, the second model explains why mutations that change the rate of deactivation often produce similar effects on the rate of recovery from desensitization (14, 33, 34).

Independent of gating model, the entry into desensitization is associated with modification of the D1-D1 interface (fig. S12) (18, 20, 3537). One possible modification is represented by structures of the S729C and G725C cross-linked isolated LBDs (18) where the D1-D1 interface is ruptured. However, K493C cross-linking does not affect desensitization (Fig. 4C) and argues against these structures representing the desensitized state of the intact receptor. Alternatively, the D1-D1 interface modification might be a rotation of the D1 lobes relative to each other that does not change the distance between K493 lysines but introduces relative displacement of pairs of other residues at the D1-D1 interface. Correspondingly, mutations like L483Y (28) or S497C (Fig. 4C and fig. S11) or positive allosteric modulators like CTZ (10, 29) would block desensitization by imposing constraints on the D1-D1 interface rearrangement.

Supplementary Materials

www.sciencemag.org/content/345/6200/1070/suppl/DC1

Materials and Methods

Figs. S1 to S12

Tables S1 to S3

References (3850)

References and Notes

  1. Acknowledgments: We thank the personnel at beamlines X4A, X4C, X25, and X29 of the National Synchrotron Light Source and at beamlines 24-ID-C and 24-ID-E of the Advanced Photon Source. 24-ID-C and 24-ID-E are the Northeastern Collaborative Access Team beamlines, which are supported by a grant from the National Institute of General Medical Sciences (P41 GM103403) from the NIH. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. We thank L. Wollmuth and R. Kazi for help in setting up electrophysiological experiments and K. Saotome for comments on the manuscript. This work was supported by the NIH (NS083660) and the Klingenstein Foundation (A.I.S.). Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4U4F for GluA2NOW and 4U4G for GluA2ZK.
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