AMPA Receptor Ligand-binding Domain Research Articles

Mechanism of AMPA Receptor Modulation by Gamma-8

Fast excitatory neurotransmission is mediated by ionotropic glutamate receptor (iGluR). AMPA-subtype ionotropic glutamate receptors (AMPARs) are associated with auxiliary subunits (transmembrane AMPA receptor regulatory proteins (TARPs), cornichons, cysteine knot proteins and GSG1); which regulate AMPAR assembly, trafficking, gating, and pharmacology. Of particular interest is the interactions with gamma-8 which results in re-sensitization of the currents where the transmembrane channel in the receptor initially undergoes closure due to desensitization but reopens at longer time and remain open in the continued presence of agonist. Here we have investigated the mechanism underlying this resensitization process using single molecule FRET and single channel functional recordings. Specifically we have determined changes at the AMPA receptor ligand binding domain dimer interface in presence of gamma-8 and compared it to the changes at this site observed in the presence of cyclothiazide, a drug that stabilizes the open channel state of the receptor. These studies show that while the overall states populated are similar in the two cases, the kinetics of transitions between states are different, with activation barrier between transitions being higher in the presence of gamma-8 . The single channel current recordings reflected these slower kinetics in the presence of gamma-8 and also showed very rapid channel openings and closing which were no reflected at the dimer interface suggesting that these may be localized changes at the transmembrane segments.

Effects of T686A Mutation on the Structural Stability of the AMPA Receptor Ligand-Binding Domain

Effects of T686A Mutation on the Structural Stability of the AMPA Receptor Ligand-Binding Domain

Stargazin Modulation of AMPA Receptors

Fast excitatory synaptic signaling in the mammalian brain is mediated by AMPA-type ionotropic glutamate receptors. In neurons, AMPA receptors co-assemble with auxiliary proteins, such as stargazin, which can markedly alter receptor trafficking and gating. Here, we used luminescence resonance energy transfer measurements to map distances between the full-length, functional AMPA receptor and stargazin expressed in HEK293 cells and to determine the ensemble structural changes in the receptor due to stargazin. In addition, we used single-molecule fluorescence resonance energy transfer to study the structural and conformational distribution of thereceptor and how this distribution is affected bystargazin. Our nanopositioning data place stargazin below the AMPA receptor ligand-binding domain, where it is well poised to act as a scaffold to facilitate the long-range conformational selection observations seen in single-molecule experiments. These data support a model of stargazin acting to stabilize or select conformational states that favor activation.

Conformational Dynamics of the GluK2 Ligand-Binding Domain

The kainate family of ionotropic glutamate receptors (GluK) mediates neurotransmission in pre- and post-synaptic neurons. Closure of the ligand-binding domain (LBD) in response to agonist binding is a necessary conformational transition to gate the ion channel. Previously computed free energy landscapes for iGluR LBDs revealed subtype-dependent conformational differences between AMPA and NMDA iGluR families, which may help to explain functional differences between iGluR subtypes. Here we report free energy landscapes, or potentials of mean force (PMFs), for the GluK2 LBD in apo and glutamate-bound states. GluK2 PMFs reveal both apo and glutamate-bound LBDs preferentially access a closed-cleft conformation, consistent with a conformational selection mechanism for binding. These results are also consistent with PMFs for NMDA but not AMPA receptor LBDs. Additionally, the open-cleft conformation is more accessible to the GluK2 apo state than the glutamate-bound state, consistent with previous results for both NMDA and AMPA receptors. Small-angle X-ray scattering profiles for the apo- and glutamate-bound states calculated from a Boltzmann-weighted ensemble of conformations are compared with experimental SAXS profiles. Finally, structural and biochemical properties of GluK2 in its apo state are probed using proteolytic protection assays, circular dichroism, and multi-angle light scattering.

Stargazin promotes closure of the AMPA receptor ligand-binding domain.

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) markedly enhance AMPAR function, altering ligand efficacy and receptor gating kinetics and thereby shaping the postsynaptic response. The structural mechanism underlying TARP effects on gating, however, is unknown. Here we find that the prototypical member of the TARP family, stargazin or γ-2, rescues gating deficits in AMPARs carrying mutations that destabilize the closed-cleft states of the ligand-binding domain (LBD), suggesting that stargazin reverses the effects of these mutations and likely stabilizes closed LBD states. Furthermore, stargazin promotes a more closed conformation of the LBD, as indicated by reduced accessibility to the large antagonist NBQX. Consistent with the functional studies, luminescence resonance energy transfer experiments directly demonstrate that the AMPAR LBD is on average more closed in the presence of stargazin, in both the apo and agonist-bound states. The additional cleft closure and/or stabilization of the more closed-cleft states of the LBD is expected to translate to higher agonist efficacy and could contribute to the structural mechanism for stargazin modulation of AMPAR function.

NMDA and AMPA Receptor Ligand-Binding Domains Exhibit Subtype-Specific Conformational Propensities

The NMDA receptor family of ionotropic glutamate receptors (iGluRs) is formed by assemblies of GluN1, GluN2, and GluN3 subunits. GluN1 and GluN3 bind glycine, whereas GluN2 binds glutamate. Crystal structures of the GluN1 and GluN3A ligand-binding domains (LBDs) in their apo states reveal open- and closed-cleft conformations, respectively. Computed conformational free energy landscapes for GluN1, GluN2A, and GluN3A LBDs reveal that the apo-state LBDs sample both open- and closed-cleft conformations, suggesting a conformational selection mechanism for agonist binding.

The delta subfamily of glutamate receptors: characterization of receptor chimeras and mutants

The delta receptors, GluD1 and GluD2, are regarded as a subfamily of the ionotropic glutamate receptors solely because of sequence homology. While they play important roles in cerebellar function and high-frequency hearing and appear to serve structural functions at synapses, ligand-gated ion channel function has not been observed. However, we have previously shown that GluD2 can form functional ion channels when grafted with the ligand binding domain of a kainate receptor. In this study, we characterized this chimera as well as additional rat delta receptor chimeras and point mutants in more detail. We found that the kainate receptor ligand binding domain renders GluD1 functional as well, and GluD2 becomes a functional ion channel also when provided with an AMPA receptor ligand binding domain. Point mutations indicate that the GluD2 ion pore operates similarly but not identically to that of AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and kainate receptors. GluD2 mutated at a conserved arginine within the linker region connecting the ligand binding domain to the ion pore domain displays spontaneous currents that occur in the absence of agonists and are inhibited by agonist application - a behavior reminiscent of that of the previously characterized lurcher mutant. Using our chimeric approach, we provide evidence that this inhibition of spontaneous currents by agonists may be caused by desensitization. Our results show that delta receptors have functional gating machineries and ion permeation pathways similar but not identical to those of AMPA and kainate receptors, while the key differences seem to be located within the ligand binding domain.

Rebuttal from David M. MacLean

The main effects of the transmembrane AMPA receptor (AMPAR) auxiliary proteins (TARPs) on AMPAR gating are slowing of deactivation and desensitization, increasing peak Popen and speeding of recovery from desensitization (Priel et al. 2005; Tomita et al. 2005). All of these observations can be reproduced by increasing the rate of recovery from desensitization, γ, and by one of two additional means: either increasing the open rate, β (as proposed by Dr Howe, 2013), or reducing the cleft opening rate, CO, and desensitization rate, δ (as suggested by myself, MacLean, 2013). Moreover, increasing the channel opening rate or decreasing the cleft open rate both result in the reported substantial left-shift in steady-state glutamate EC50 values produced by TARPs (Priel et al. 2005; Tomita et al. 2005; Kott et al. 2007) as well as the smaller left-shift in peak EC50 values (Morimoto-Tomita et al. 2009). Therefore, such concentration–response data are not helpful in distinguishing an effect on gating transitions from an effect on pre-gating transitions such as stabilizing closed-cleft states. However, peak inhibition curves using NBQX and CNQX may provide some support for the hypothesis that TARPs affect pre-gating transitions by promoting states with closed ligand-binding domains (LBDs). The LBDs of AMPARs exist in multiple closed-cleft conformations, both when the agonist is bound and in the apo state (Landes et al. 2011). If the presence of TARPs causes AMPAR LBDs to adopt more closed conformations, then even in the apo state the binding cleft would be, on average, slightly more shut. Consequently, larger ligands such as NBQX or CNQX would have slower binding rates since there would be fewer favourable collisions between these compounds and the more closed LBDs. Such slowed binding rates of larger antagonists would result in right-shifts of peak inhibition curves and this is precisely what is observed experimentally. The peak response IC50 values obtained using rapid perfusion on outside-out patches for both CNQX and NBQX are right-shifted approximately 2-fold by stargazin (MacLean & Bowie, 2011). While this observation is consistent with TARPs stabilizing more closed LBD conformations, more compelling evidence is needed. Ultimately, to discriminate between these mechanisms, direct measurements of both the degree of LBD closure and coupling between LBD closure and channel opening will be required.

The loss of an electrostatic contact unique to AMPA receptor ligand binding domain 2 slows channel activation.

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.

AMPA Receptor Ligand Binding Domain Mobility Revealed by Functional Cross Linking

Glutamate receptors mediate the majority of excitatory synaptic transmission in the CNS. The AMPA-subtype has rapid kinetics, with activation, deactivation and desensitization proceeding on the millisecond timescale or faster. Crystallographic, biochemical, and functional studies suggest that GluR2 Cys mutants which form intermolecular disulfide cross-links between the lower D2 lobes of the ligand binding cores can be trapped in a conformation that represents the desensitized state. We used multi-channel rapid perfusion techniques to examine the state dependence of cross-linking in these mutants. Under reducing conditions, both wild-type GluR2 and the G725C and S729C mutants have normal activation and desensitization kinetics, but the Cys mutants can be efficiently trapped in nonconducting states when oxidized. In contrast the I664C mutant is only partially inactivated under oxidizing conditions. For S729C, disulfide cross-links form rapidly when receptors are desensitized in the presence of glutamate, but receptors also become trapped at rest, in the absence of agonist. We assessed such spontaneous trapping in various conditions, including CNQX, a competitive antagonist; kainate, a weak partial agonist; or when desensitization was blocked by the L483Y mutation that stabilizes the D1 dimer interface. These experiments suggest that trapping in the absence of glutamate is due to two motions: Spontaneous breaking of the D1 dimer interface and hyperextension of the lower lobes of the ligand binding core. These data show that the glutamate binding domains are surprisingly mobile in the absence of ligand, which could influence receptor activity in the brain.

Stability of ligand-binding domain dimer assembly controls kainate receptor desensitization

AMPA and kainate receptors mediate fast synaptic transmission. AMPA receptor ligand-binding domains form dimers, which are key functional units controlling ion-channel activation and desensitization. Dimer stability is inversely related to the rate and extent of desensitization. Kainate and AMPA receptors share common structural elements, but functional measurements suggest that subunit assembly and gating differs between these subtypes. To investigate this, we constructed a library of GluR6 kainate receptor mutants and directly measured changes in kainate receptor dimer stability by analytical ultracentrifugation, which, combined with electrophysiological experiments, revealed an inverse correlation between dimer stability and the rate of desensitization. We solved crystal structures for a series of five GluR6 mutants, to understand the molecular mechanisms for dimer stabilization. We demonstrate that the desensitized state of kainate receptors acts as a deep energy well offsetting the stabilizing effects of dimer interface mutants, and that the deactivation of kainate receptor responses is dominated by entry into desensitized states. Our results show how neurotransmitter receptors with similar structures and gating mechanisms can exhibit strikingly different functional properties.

Purification and crystallization of a non-GluR2 AMPA-receptor ligand-binding domain: a case of cryo-incompatibility addressed by room-temperature data collection

Glutamate is the major excitatory neurotransmitter in the brain. Among the cognate ionotropic glutamate receptors, the subfamily selective for AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) is responsible for most fast excitatory synaptic signaling and plays key roles in synaptic plasticity. AMPA receptors (AMPA-Rs) have also been implicated in a number of neurological disorders. To investigate subunit-specific differences in the ligand binding and activation of AMPA-Rs, the GluR4 AMPA-R ligand-binding domain (LBD) was crystallized in complex with full and partial agonists. This is the first non-GluR2 AMPA-R LBD available for structural analysis. Standard cryoprotection protocols yielded high-resolution diffraction from flash-cooled crystals of the complex with the full agonist glutamate. However, for cocrystals with the partial agonist kainate, systematic screening and optimization of cryoprotection conditions yielded at best mosaic, weak diffraction at 100 K. In contrast, room-temperature data collection from capillary-mounted kainate cocrystals exhibited reproducible diffraction to better than 3 A resolution. Together, these crystals lay the foundation for a structural comparison of LBD-agonist interactions in distinct AMPA-R subunits.

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain.

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.