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. 2016 Sep 22;537(7621):567-571.
doi: 10.1038/nature19352. Epub 2016 Aug 31.

Structural Basis of Kainate Subtype Glutamate Receptor Desensitization

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Free PMC article

Structural Basis of Kainate Subtype Glutamate Receptor Desensitization

Joel R Meyerson et al. Nature. .
Free PMC article

Abstract

Glutamate receptors are ligand-gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases. In both the resting and desensitized states of AMPA and kainate receptor subtypes, the ion channels are closed, whereas the ligand-binding domains, which are physically coupled to the channels, adopt markedly different conformations. Without an atomic model for the desensitized state, it is not possible to address a central problem in receptor gating: how the resting and desensitized receptor states both display closed ion channels, although they have major differences in the quaternary structure of the ligand-binding domain. Here, by determining the structure of the kainate receptor GluK2 subtype in its desensitized state by cryo-electron microscopy (cryo-EM) at 3.8 Å resolution, we show that desensitization is characterized by the establishment of a ring-like structure in the ligand-binding domain layer of the receptor. Formation of this 'desensitization ring' is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo-four-fold symmetric arrangement of the ligand-binding domains, illustrating subtle changes in symmetry that are important for the gating mechanism. Disruption of the desensitization ring is probably the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Desensitized GluK2 imaging and structure determination
a, b, Representative cryo-EM image of GluK2EM solubilized in DDM-CHS and bound by 2S,4R-4-methylglutamate (a), with the corresponding image power spectrum and CTF estimate showing signal beyond 3 Å resolution (b, solid and dotted lines, respectively). The defocus value for the image is 1.5 μm. In panel (a) particles are highlighted with circles, and image binning at 4x and uniform level adjustments were used to make particles apparent. Scale bar is 500 Å. c, Subset of selected two-dimensional class averages. d, FSC curve with reported resolution of 3.8 Å at the 0.143 crossing. e, Structure of agonist bound GluK2EM colored according to local resolution, shown at three progressively increasing contours.
Extended Data Figure 2
Extended Data Figure 2. Desensitized GluK2 transmembrane and glycosylation features
a, b, Cryo-EM density for resolved S1-M1 (a) and M3-S2 (b) linkers and TM helices, displayed with Cα trace of the atomic model. c, Representative sites with densities for complex glycans at Asn244, Asn347, and Asn399.
Extended Data Figure 3
Extended Data Figure 3. Reconstruction of desensitized GluK2 without computational symmetry
a, Cryo-EM density map for the reconstruction of agonist bound GluK2 without imposition of computational symmetry, and colored according to local resolution. b, FSC curve with reported resolution of 4.4 Å at the 0.143 crossing. c, Segmentation of individual GluK2 chains of the asymmetric reconstruction. The density map segmentation is shown fitted with a trace representation of the model displayed in Fig. 1b.
Extended Data Figure 4
Extended Data Figure 4. Inhibition of GluK2EM by LY466195 and LBD crystal structures for agonist and antagonist complexes
a, Crystal structure for the GluK2EM isolated LBD dimer assembly complex with 2S,4R-4-methylglutamate; the upper/lower lobes for the two subunits are colored orange/pale yellow and teal/pale cyan respectively; the dashed line indicates the separation of the lower lobes measured as the distance between the Cα positions of Ile637. b, Crystal structure for the GluK2EM isolated LBD dimer assembly complex with LY466195 illustrating the large decrease in separation of the lower lobes compared to the agonist complex. Coloring is the same as in (a). c, Responses to 100 μM glutamate recorded under two electrode voltage clamp for GluK2EM (top) and wild type GluK2 (bottom); the initial response to glutamate recorded after prior application of 300 nM LY466195 showed nearly complete block for GluK2EM with no change in amplitude for wild type. d, Concentration dependence for inhibition of GluK2EM by LY466195 yielded an IC50 of 30 nM.
Extended Data Figure 5
Extended Data Figure 5. Imaging and structure of GluK2EM bound by antagonist LY466195
a, b, Representative cryo-EM image of GluK2EM solubilized in DDM-CHS and bound by LY466195 (a), with the corresponding image power spectrum and CTF estimate showing signal beyond 6 Å resolution (b, solid and dotted lines, respectively). The defocus value for the image is 2.7 μm. In panel (a) particles are highlighted with circles, and image binning at 4x and uniform level adjustments were used to make particles apparent. Scale bar is 500 Å. c, Subset of selected two-dimensional class averages. d, Cryo-EM density map for GluA2 bound to ZK200775 (left), density map for GluK2EM bound to LY466195 (middle) and its corresponding molecular model built from ATD and LBD dimers (right). e, FSC curve with reported resolution of 11.6 Å at 0.143 crossing.
Extended Data Figure 6
Extended Data Figure 6. ATD-LBD interface of desensitized GluK2
a, Desensitized GluK2 shown in surface representation with ATD-LBD interfaces highlighted. b, Top down view of LBD layer shown with perspective indicated by eye icon in (a). c, Underside of the ATD layer as viewed after peeling away from LBD layer. Dashed lines in (a) highlight where the layers are separated. In all panels, interfaces on chain A and C are in green and blue, respectively. d, Table with residues that mediate ATD-LBD interaction. e, f, Cartoon representation of LBD and ATD layers from same views as in (b) and (c), with interface residues colored to correspond with table in (d).
Extended Data Figure 7
Extended Data Figure 7. Desensitization ring residues that influence recovery kinetics
a–d, Rate of recovery from desensitization measured using twin pulse applications of 10 mM glutamate (data points show mean ± SD; fits are shown in red). a, Wild type GluK2 fit with a single exponential. b, D672R fit with the sum of two exponentials with the response for wild type shown as a black line; c, S669R D672R double mutant fit with the sum of two exponentials with the response for wild type shown as a black line. d, Time to 50% recovery in seconds. e–h, Top views of the GluK2 desensitization ring with residues found to influence recovery kinetics when mutated. Each panel shows the wild-type residue alpha carbon position as a sphere. Positions for the S669R and D672R mutations from the present study are shown in (e) and (f), respectively. The A676T and S679R positions found in previous studies,, are in (g) and (h), respectively.
Figure 1
Figure 1. Desensitized kainate receptor at 3.8 Å resolution
a, b, Cryo-EM density map (a) and atomic model (b) of 2S,4R-4-methylglutamate bound GluK2EM with each chain colored uniquely. Panel (a) has features shown from two map contours. c, Side and top view of the ATD tetramer, with cartoon highlighting 2-fold symmetry in the ATD layer. d, Top view of LBD tetramer with cartoon illustrating apparent 4-fold symmetry of the LBD layer when seen down the receptor central axis. e, Top view of the TMD with cartoon indicating domain symmetry.
Figure 2
Figure 2. Desensitization ring
a, Top view of LBD layer highlighting desensitization ring helices E and G colored magenta for AC subunits and cyan for BD subunits. Residues S669 and D672, mutated in functional experiments (g–i), are shown as sticks to highlight their positions. b, Edge-on view of desensitization ring interface with perspective indicated by eye icon in (a). c, Same perspective as in (b) but rotated 90° to show the second type of E/G interface observed in the desensitization ring. d, Illustration of hypothetical desensitization ring arrangement that would yield complete 4-fold symmetry in the LBD layer. The G helices are shown on top layer, and E helices on bottom layer. e, Illustration of the experimentally observed desensitization ring arrangement which is staggered and has 2-fold symmetry. f, The BD LBDs (left) are less inclined than the AC LBDs (right). This tilting of the AC domains elevates their E/G helices to adopt the upper position in the staggered configuration. Helix B of each LBD is highlighted in yellow as a reference to show domain tilt. g, Response to 10 mM glutamate for the D672R mutant, with the onset of desensitization fit by a single exponential of time constant 1.5 ms (red line). The upper trace shows the open tip junction current recorded at the end of the experiment. h, Recovery from desensitization, recorded from the same patch, for twin pulse applications of glutamate. The upper trace shows the command to the piezo stack. i, Recovery from desensitization for the D672R mutant (data points show mean ± SD, n=12) and wild type GluK2 (dashed line) fit with exponential functions.
Figure 3
Figure 3. Desensitized state ion channel
a–c, Selected sidechain densities with atomic model for M1 (a), M3 (b), and M4 (c) helices. d, The ion channel with surface modeling of the channel. Regions with pore radius less than 1.15 Å are red, water accessible parts with radius between 1.15 Å and 2.30 Å are green, and wide areas with pore radius greater than 2.30 Å are blue. e, Plot of pore radius as a function of channel position with desensitized GluK2 (black), apo GluA2 (blue, PDB ID: 4U2P), antagonist-bound resting GluA2 (orange, PDB ID: 3KG2), and pre-activated GluA2 (magenta, PDB ID: 4U5B). Residue labels on right hand Y-axis mark the position of the respective channel forming side chains.
Figure 4
Figure 4. LBD-TM linkers mediate channel closing and LBD reorganization
a, Comparison of the regions encompassing the M3, M3-S2 linkers, and E helices for AC (magenta) and BD (cyan) chains of desensitized state GluK2EM. b, The vertical rise in Cα position as a function of residue number for chains AC (magenta) versus BD (cyan). c, Measurement of how Cα positions on desensitized state GluK2 chains A, C, B, and D deviate from corresponding positions on resting state GluA2cryst which was used as a reference. Line coloring is the same as in panel (b). Dotted lines indicate AB chains, with solid lines for CD chains. The magenta traces show low deviation, reflecting the similarity between these regions of the AC subunits of desensitized GluK2 and the resting state GluA2 structure used as a reference (PDB ID: 3KG2). The cyan trace begins to deviate significantly at the circular markers corresponding to R632 (R628) GluK2 (GluA2). This reflects the dramatic difference in BD linker arrangements between the resting and desensitized states. d, The desensitized GluK2 channel with the region considered in (c). M3 helices and M3-S2 linkers are colored as in other panels, with pre-M1, M1 and M4 regions colored in yellow. Spheres mark residue R632 on all four TM chains. e, Schematic view of structural rearrangements that are involved in transition from resting to activated to desensitized states of glutamate receptors. Supplementary Video 1 shows an animation of the gating cycle.

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