Structural insights into kainate receptor desensitization

Abstract

Kainate receptors (KARs) belong to the ionotropic glutamate receptor (iGluR) family and play critical roles in mediating excitatory neurotransmission and regulating neurotransmitter release. Receptor desensitization is a critical factor for regulating the strength of synaptic transmission. Notwithstanding their overall structural similarity to AMPA receptors, KARs exhibit a desensitized conformation that is distinct from that of most other iGluRs. Despite extensive studies on KARs, a fundamental question remains unresolved: why do KARs require large conformational changes upon desensitization? Here we show cryo-electron microscopy structures of GluK2 containing double cysteine mutations, captured in non-active and various desensitized conformations. In the shallow-desensitized conformation, two cysteine crosslinks stabilize the receptors in a conformation resembling the typical desensitized state of non-KAR iGluRs. Our patch-clamp recordings and fluctuation analysis suggest that KARs in the shallow-desensitized state remain ion-permeable. This finding indicates that the lateral rotational movement of the KAR ligand-binding domains is critical for complete channel closure and stabilization of the fully desensitized receptor. Overall, this study elucidates the mechanism and conformational dynamics of KARs during desensitization.

Fig. 1. Functional and structural characterization of GluK2 K676C/N802C.

A Representative whole-cell patch-clamp trace showing the response of GluK2 WT or GluK2 K676C/N802C to a 20-s application of 1 mM glutamate in the presence and absence of BPAM344 (BPAM). Arrows indicate the maximum peak current (I_peak) and the steady-state current (I_ss). B Quantification of percentage desensitization for currents evoked by 1 mM glutamate in GluK2 (dark gray; n = 10), and the K676C/N802C mutant (light gray; n = 7), and for currents evoked by 1 mM glutamate plus 500 μM BPAM344 in GluK2 WT (green; n = 6) and the K676C/N802C mutant (cyan; n = 10) (C) Ratios of peak current amplitudes (I_peak) for GluK2 WT (left; n = 6), and K676C/N802C (middle; n = 10), and of steady-state current amplitude (I_ss) for GluK2 K676C/N802C (right; n = 10), measured in the presence and absence of BPAM344. For B and C, black circles denote independent biological replicates. Data are mean ± SD; whiskers indicate the standard deviation. Statistical significance was calculated using a two-sided two-sample t-test, with significance assumed if P < 0.05; the exact P-values are shown in the figure. D Cryo-EM map and model of GluK2 K676C/N802C complexed to glutamate and BPAM344 in a non-active state. N-glycans are highlighted in yellow. E The LBDs shown in views parallel (top) and perpendicular (bottom) to the membrane. BPAM344 and glutamate are depicted as space-filling models, coloured pink and cyan, respectively. Disulfide bonds formed between K676C and N802C, and the BPAM344 binding sites, are indicated by black and cyan dashed boxes, respectively. F Schematic diagram illustrating LBD with cysteine crosslinks, which are indicated by black lines (i) and (ii). In the diagram, glutamate is represented by a pink circle, and BPAM344 is represented by a cyan circle. G Comparison of the apo GluK2 LBD (PDB 9CAZ) and the glutamate-bound GluK2 LBD in the non-active LBD conformation. The D1 lobe is superimposed, and the rotation angles to align the D2 residues were calculated. The glutamate-bound LBD exhibits a 20° closure relative to the apo conformation. H Chemical structure of BPAM344. I Close-up view of the BPAM344 binding site at the AD subunit interface. J Cryo-EM densities for disulfide bonds between K676C and N802C in AB and CD subunits. Source data are provided as a Source Data file.

Fig. 2. Structural mechanism of GluK2 upon activation and desensitization.

A A top-down view of the cryo-EM reconstructions and models for the BPAM344- and glutamate-bound GluK2 LBD in its non-active conformation. The distances at the LBD dimer–dimer interface, measured between S700 on the AC subunits and E788 on the BD subunits, are highlighted in red and blue, respectively. B Cryo-EM reconstructions and models of the LBD dimers in the non-active state, viewed perpendicular to the membrane. The centers of mass (COMs) for the D1 and D2 lobes are represented by blue and green spheres, respectively. The COM distances between the D1 and D2 lobes of the AD subunits (indicated by circles) are displayed below the models. The distances between the Cα atoms of P773 and S670 within the AD subunits are also presented. Arrows denote bi-lobe closure. C, D A top-down and side views of the BPAM344-, glutamate-, and concanavalin A-bound GluK2 LBD in the open state (PDB 9B36). E Comparison of LBD–TM3 linkers and TM3 formation in BPAM344-bound, agonist-unbound GluK2 (PDB 8FWS) and non-active GluK2. S670 is shown as spheres in corresponding colours for the non-active state and in grey for BPAM344-bound GluK2. Locations of the TM3 gating hinge at A656 (subunit BD) and E662 (subunit AC) are highlighted in red. The dotted line indicates the positions of pore-lining residues: (1) M664, (2) T660, (3) A656, and (4) T652. (2’) E662, which serves as the gating hinge, is also highlighted. F Comparison of LBD–TM3 linkers and TM3 formation in non-active GluK2 and BPAM344-, ConA- and glutamate bound open GluK2 structure (PDB 9B36). The locations of the gating hinge at L655 in the A–D subunits of the open GluK2 structure are highlighted in grey. Arrows indicate the rearrangement of TM3. G Pore profile in the non-active structure. Pore-delineating dots are coloured according to pore radius: red for regions with a radius <1.1 Å and grey for regions with a radius >1.1 Å, which would allow passage of a dehydrated ion (1.1 Å for calcium). H The pore radius for the non-active (red) and open/active (PDB code, 9B36, grey) conformations, calculated using HOLE. Source data are provided as a Source Data file.

Fig. 3. Structures of GluK2 in desensitized states.

A Three-dimensional (3D) cryo-EM reconstructions of glutamate-bound GluK2 K676C/N802C in desensitized conformations. B Top views of glutamate-bound GluK2 K676C/N802C, focusing on the LBD layers. The in-plane rotation angles of the LBDs are indicated. Disulfide bonds formed between the two LBD dimers are highlighted in red dotted boxes. C Close-up view of the disulfide bonds between K676C and N802C at the CD subunit interface (left) and the AC subunit interface (right) in the shallow-desensitized conformation. D Top view of the LBD layer in the deep-desensitized conformation, highlighting helices E and G, which form the desensitization ring. The view is parallel to the membrane, with colouring consistent with A. E The desensitization ring formed in three deep-desensitized conformations, viewed perpendicular to the membrane. The in-plane rotation of the LBD shown in B shifts the LBD–TM3 linker, positioning it above the ion channel pore formed by the TM3 helices.

Fig. 4. GluK2 stabilized in a shallow-desensitized-like conformations resembles the desensitized conformation of AMPARs.

A, B Comparison between the non-active and shallow-desensitized conformations, viewed parallel (A) and perpendicular (B) to the membrane. Black arrows indicate the degree of LBD domain rotation. C, D Structural comparison of LBD layers in GluK2 K676C/N802C in the shallow-desensitized state and GluA2 AMPAR in the desensitized state (PDB 7RYZ), viewed parallel (C) and perpendicular (D) to the membrane. Distances between S700 in the AC subunits and E788 in the BD subunits are shown in red and blue, respectively. The locations of the centers of mass (COMs) of D1 and D2 are indicated by blue and green spheres, respectively, with the COM distances between D1 and D2 of the AD subunit of the LBDs displayed below. Arrows indicate D1 lobe rotations compared to the non-active (KAR) or active (AMPAR) states. Distances between the Cα atoms of P773 (GluK2)/P745 (GluA2) and S670 (GluK2)/S635 (GluA2) in the AD subunits are indicated. E Comparison of LBD-TM3 linkers and TM3 helices in non-active and shallow-desensitized conformations. S670 is shown as spheres in corresponding colours for the shallow-desensitized conformation and in gray for the non-active conformation. The cross-dimer distances between the Cα atoms of S670 are indicated. Residues forming the ion channel pore are displayed as sticks. The locations of the TM3 gating hinge at T660 (subunit BD) and M664 (subunit AC) in the shallow-desensitized conformation are highlighted in red. The shallow-desensitized conformation contains an additional half-turn of helices at T660 compared to the non-active conformation. F Comparison of LBD–TM3 linkers and TM3 formation in shallow- and deep-desensitized GluK2. In the deep-desensitized conformation, the ion channel is sealed at M664, completely closing the ion channel pore, in contrast to the shallow-desensitized conformation, which has a kink at T660 in the BD subunits. G Extracellular views of the ion channel in the shallow-desensitized and deep-desensitized states, showing M664 positioned at the top of the gate region as sticks (top). The schematic illustration compares these two desensitized states, highlighting how M664 partially or completely seals the pore (bottom).

Fig. 5. Ion channel pore of desensitized GluK2 KAR.

A Permeation pathway dictating the pore diameter of GluK2 K676C/N802C in five desensitized states. Pore-delineating dots are colour-coded based on radius: red indicates regions with a radius <1.1 Å, while grey denotes regions >1.1 Å. B, C Pore profile comparisons. A comparison of the pore profiles between the shallow-desensitized and intermediate states (B), and a comparison of the pore profiles among three distinct deep-desensitized states (C). D Permeation pathway of the desensitized GluA2 AMPAR (PDB code 7RYZ). The pore radius is colour-coded according to the scheme in A. E A pore profile comparison between GluK2 K676C/N802C in the shallow-desensitized conformation and the desensitized GluA2 AMPAR (PDB code, 7RYZ). Source data are provided as a Source Data file.

Fig. 6. Functional characterization of GluK2 K676C/N802C receptor.

A Representative whole-cell patch-clamp recordings of GluK2 WT (left) and GluK2 K676C/N802C (right) in response to 0.001–50 mM glutamate. The black bar represents the glutamate application. B Quantification of current density (pA/pF) at the peak current for GluK2 WT (gray, n = 6) and K676C/N802C (blue, n = 9) activated by 10 mM glutamate. A two-sided two-sample t-test was performed, and no significant differences were observed (P = 0.324). C Glutamate dose-response curves for GluK2 WT (n = 12) and K676C/N802C (n = 8), normalized to the maximal response (50 mM). Glutamate was applied for 1 s, and the peak current was measured at each concentration. All concentrations were tested in the same cell. D EC₅₀ values for glutamate activation of GluK2 WT (gray, n = 7) and K676C/N802C (blue, n = 5). A two-sided two-sample t-test was performed, and no significant differences were observed (P = 0.471). E Ratio of steady-state to peak current (I_steady-state/I_peak) (50 mM, n = 6; 10 mM, n = 10; 1 mM, n = 6; 0.01 mM, n = 6; 0.001 mM, n = 6). The values of the steady-state current were measured at the end of the activation period. F Representative current traces of GluK2 WT (black) and K676C/N802C (blue) during a two-pulse protocol, showing the interval between glutamate exposures ranging from 50 ms to 20 s. G Recovery from desensitization at increasing inter-sweep intervals, fitted with the Hodgkin-Huxley (I(t)=[(I_max^1/m-(I_max^1/m-I₀^1/m)) ×e^-t/τ]^m for GluK2 WT and two-term exponential functions for GluK2 K676C/N802C. The K676C/N802C mutant recovers faster than WT. H Time constant (τ) of recovery from desensitization, showing a significant reduction in τ for the K676C/N802C mutant (P < 0.0001, two-sided two-sample t-test). The data for the WT were obtained from the fit in (G), and for K676C/N802C, a mean τ was calculated using τ_mean = ((A1 τ1 + A2 τ2) / (A1 + A2)). Biologically independent measurements: n = 11 for GluK2 WT and n = 10 for K676C/N802C. Black circles represent biological independent measurements. Data are presented as mean ± SD; whiskers indicates the Standard Deviation (SD). Statistical analysis was performed by applying a two-sided two-sample two-tailed t-test. Exact P-values are shown in the figure; significance is assumed if P < 0.05. Source data are provided as a Source Data file.

Fig. 7. Effects of DTT and BPAM344 modulation on the steady-state current of GluK2 K676C/N802C.

A Representative whole-cell patch-clamp recordings of GluK2 WT (left) and GluK2 K676C/N802C (right) activated by 10 mM glutamate before (black) and after (red) treatment with 5 mM DTT for 7 min. B I_ss/I_peak ratio in GluK2 K676C/N802C (n = 7), showing a significant reduction in steady-state current (I_ss) (P < 0.0001, paired t-test). C Recovery from desensitization at increasing inter-sweep intervals after treatment with 5 mM DTT. The recovery kinetics of GluK2 K676C/N802C post-DTT treatment were fitted with the Hodgkin-Huxley (I(t)=[(I_max^1/m-(I_max^1/m-I₀^1/m)) ×e^-t/τ]^m). D Time constant (τ) of recovery from desensitization, showing that DTT treatment restores the recovery kinetics of GluK2 K676C/N802C (n = 5) to a level comparable to WT (n = 9) (P = 0.258, two-sided two-sample t-test). E Representative whole-cell responses to 10 mM glutamate (200 ms, −70 mV; black bar) of GluK2 WT (left, average current in black, n = 60 individual sweeps in gray) or GluK2 K676C/N802C (right, average current in blue, 100 individual sweeps in gray). Inset: Current-variance relationship, with WT data fitting a parabolic function (dotted line), whereas the K676C/N802C mutant does not follow this trend. F Whole-cell recordings of GluK2 K676C/N802C in response to 10 mM glutamate in the absence (blue) and presence (green) of 500 µM BPAM344. Right: Representative noise traces at the end of glutamate application ( ~ 10 s). Background noise was taken from the current recording before glutamate application. G Variance-mean current relationship for individual cells (n = 6), indicating an increase in channel variance-current relationship in the presecen of BPAM344. Data were obtained from currents shown in panel F. H Unitary conductance determined by variance-mean analysis (n = 6), showing no significant difference with and without BPAM344 (P = 0.280, two-sided paired t-test). I Estimated number of open channels, showing a significant increase after BPAM344 application (P < 0.0001, two-sided paired t-test). The black circles represent biologically independent measurements. Data for glutamate and BPAM344 conditions were obtained from the same cell. Data are presented as means, with whiskers representing standard deviation (SD). Statistical significance is indicated as NS (not significant), and p-values are provided in the figure legend or shown in the figure. Source data are provided as a Source Data file.

Fig. 8. Schematic representation of conformational rearrangements during activation and desensitization.

This schematic model details how conformational changes within the LBD layer regulate ion channel permeation during desensitization. The LBD tetramer conformation for each state is depicted within a circle. The position of the gating kink varies across the non-active, open, and desensitized states. The shallow-desensitized state is specifically observed when the LBD dimer-of-dimers conformation is stabilized by cysteine cross-linking. While the shallow-desensitized conformation exhibits incomplete closure at the top of the channel gate, the pore of the deep-desensitized states is tightly and stably sealed by the T660 and M644 constriction sites. For comparison, the previously determined open state (PDB code: 9B35) is shown. The apo state conformation is represented by two structures (PDB codes: 9CAZ and 8FWS).