Structural basis for activation and conformational plasticity of the GluA4 AMPA receptor

Domain: chemistry
Published: 2026-02-09
Editorial outcome: accept

Abstract
AMPA-subtype glutamate receptors (AMPARs) mediate excitatory synaptic transmission. AMPAR ion channels exhibit multiple subconductance states that tune neuronal responses to glutamate. GluA4 is the rarest subunit in the brain but is enriched in interneurons and the cerebellum. Rising evidence points to GluA4 AMPARs in the development of neurological diseases, but the structural mechanisms of GluA4 function remain enigmatic. Here, we show the distinct features of GluA4 that tune AMPAR function. We find that GluA4 AMPARs have a canonical “Y” shaped architecture where local dimer pairs are domain-swapped between the amino terminal domain (NTD) and ligand binding domain (LBD), both of which comprise the extracellular domain. All four LBDs are glutamate bound yet open the GluA4 ion channel by asymmetric hinging in all four channel helices. We observe that the glutamate-saturated LBD has conformational plasticity, which tunes the ion channel gate below. These data provide a framework for understanding channel subconductance, outline the distinct properties of GluA4, expand our understanding of conformational plasticity in AMPARs, and will inform therapeutic design.

Introduction
Fast, excitatory neurotransmission in the vertebrate brain is principally mediated through AMPA-subtype glutamate receptors (AMPARs) – ligand-gated ion channels that open a cation-selective pore in the neuronal membrane in response to binding the neurotransmitter glutamate1,2. Transmission through AMPARs is thought to mediate essential processes in cognition, and plasticity of AMPARs alters synaptic strength to mediate the most well-characterized physiological correlates of neurological memory. Dysfunction in AMPAR signaling is increasingly implicated in diverse neurological and psychiatric diseases, including Alzheimer’s dementia, intellectual disability, and schizophrenia1.

Accordingly, there is intense interest in understanding the mechanisms by which AMPARs function and are regulated at synaptic sites.

AMPARs are tetrameric assemblies formed by four pore-forming subunits from the GluA family of proteins (GluA1–4). AMPAR subunits are unevenly expressed among neuronal cell types and across brain regions3, and the subunit composition of AMPARs at synapses can be dynamically regulated during certain forms of AMPAR plasticity4. AMPARs bearing the widely expressed GluA2 subunit are rendered impermeable to calcium and structures of native AMPARs purified from brain suggest that GluA2 maintains a privileged structural relationship within the receptor5. However, increasing evidence suggests a critical role for calcium-permeable AMPARs (CP-AMPARs) in the brain6,7, which either lack GluA2 or include rare calcium-permeable forms of GluA28.

Certain cell types in the brain, such as disease-associated Parvalbumin-expressing interneurons (PVINs), and principal cells in the Medial Nucleus of the Trapezoid Body (MNTB), express relatively little of the GluA2 subunit and instead express relatively high levels of the poorly understood calcium-permeable GluA4 subunit9,10, which exhibits distinct physiological properties11–13. GluA4 homomers may be central to excitatory drive at the Calyx of Held14, and the calcium-permeability of GluA4-containing receptors is critical to the physiological properties of PVINs and altering the calcium permeability in these cells undermines their organismal function9. Plasticity of AMPARs on PVINs in cortical cultures can be mediated by GluA4, highlighting the importance of this subunit to the physiology of these cells15,16. Rare variants in the gene encoding GluA4 are increasingly found in patients with severe intellectual disability and epilepsy, highlighting the role of GluA4 in human disease17, and genetic deletion of the gene encoding GluA4 from mice produces an epileptic phenotype18,19. Other than in sparse interneuron classes, GluA4 expression is high in areas of the auditory system20–25 and the cerebellum3,11,26,27, where it likely plays specialized roles in cellular and organismal physiology14,28,29. GluA4 is expressed early in the hippocampus where it is critical for synaptic plasticity30,31. Dysfunctional GluA4 expression and signaling have also been associated with glioblastoma growth and tumor proliferation32–34. However, what mediates the distinct features of GluA4 function is enigmatic.

To address this, we used cryo-electron microscopy (cryo-EM) to solve the structure of a GluA4 homotetrameric AMPAR in complex with the auxiliary subunit TARPγ2 in the presence of glutamate and positive allosteric modulator cyclothiazide (CTZ). We identified a high degree of conformational heterogeneity in the extracellular N-terminal domain (NTD) and ligand binding domain (LBD) of the receptor that may underlie some of the distinct functional properties of the receptor and its relevance to cell types such as PVINs. These findings shed light on how GluA4-containing AMPARs differ from other calcium-permeable AMPAR subtypes and the heterogeneity in the LBD layer may provide an alternative model to the current conception of how different subconductance states of AMPARs are achieved.

Results
Purification of Functional GluA4 Homotetramers
In the cortex, GluA4 is expressed in PVINs, which also express the auxiliary subunit TARPγ235,36, which has been observed to modulate GluA4 function37. Additionally, cerebellar granule cells, which highly express GluA4 also predominantly express TARPγ2, and dysfunction of TARPγ2 in these cells produces cerebellar ataxia38–40. Therefore, to solve the structure of a GluA4 homotetrameric AMPAR we employed a fusion construct between the flip splice form of GluA4 and TARPγ2 by truncating the GluA4 sequence after the fourth transmembrane domain, followed by a short, flexible linker region, and then beginning the coding sequence for TARPγ2. TARPγ2 was truncated after the TM4 helix, and a thrombin cleavage site, GFP and polyhistidine affinity tag were added to the C-terminus immediately preceding the translational stop. This approach has been used extensively to study AMPAR/TARP complexes and a similar construct has been extensively characterized in whole cell and single-channel preparations37. Expression of this construct resulted in a large complex assessed by Fluorescence Size Exclusion Chromatography corresponding to an AMPAR tetramer (Supplementary Fig. 1a). The GluA4 peak was responsive to variations in lipid composition of the solubilization buffer, with porcine brain lipid extract demonstrating marked improvements in preserving the tetrameric assembly over DDM/CHS buffer (Supplementary Fig. 1b). While the inclusion of polar or total porcine brain lipids showed similar preservation of the tetramer, total lipid extract showed less protein lost to the void volume (Supplementary Fig. 1b, arrow) and was therefore used for purification. We therefore purified GluA4/TARPγ2 complexes by extracting the protein in a buffer containing DDM and porcine brain total lipids and then exchanging the complex into a buffer containing glyco-diosgenin (GDN; Supplementary Fig. 1c).

To test that GluA4 homotetramers were functional, we performed lipid bilayer recordings of single GluA4 channels passed into reconstituted lipid bilayers in the presence of 1 mM glutamate (Glu) and 100 μM cyclothiazide (CTZ). As compared to bilayers without protein, membranes with GluA4 homotetrameric receptors could be readily observed producing a mixture of short fluxes and prolonged openings in the presence of Glu and CTZ (Fig. 1a), consistent with currents that have been previously observed for single AMPAR channels37,41. We therefore concluded that purified GluA4/TARPγ2 complexes were capable of glutamate activation and proceeded with structural analysis of complexes purified under these conditions.

Architecture of GluA4
To examine the structure of activated GluA4, purified GluA4/TARPγ2 sample was spiked with 1 mM Glu and 100 μM CTZ before freezing, and the resulting structure was resolved via cryo-EM (Table 1, Supplementary Fig. 2). The resulting map displayed a well-resolved LBD and TMD layer, with four TARPγ2s bound around the GluA4 TMD. However, examination of the GluA4/TARPγ2 map revealed that the N-terminal Domain (NTD) was absent from our reconstruction (Supplementary Fig. 2), suggesting that the NTD of GluA4 may exhibit high conformational heterogeneity, or may even be ruptured as has been recently reported for GluA1/TARPγ3 complexes42.

The structural integrity and overall topology of AMPAR complexes depend on AMPAR subunit composition, with GluA2 driving heteromeric assembly of local dimers and contributing to the stabilization of the NTD trans-dimer interface5,43. Recent work solving the structure of GluA2-lacking AMPARs has revealed the absence of the trans-dimer interface and a ruptured NTD layer42. We therefore wondered whether the NTD was intact in our GluA4/TARPγ2 complex and endeavored to refine the NTD layer of the GluA4 particle set to see if conformational heterogeneity of the NTD layer explained its absence from our reconstruction. 3D classification of the NTD layer alone revealed multiple classes in which NTD lobes could be identified at low resolution (Supplementary Fig. 3a) and rigid-body fitting of previously determined crystal structures of the GluA4 NTD (PDB: 4GPA, Supplementary Fig. 3b)44, allowed for geometric analysis of the NTD layer. Density ascribable to four NTDs was observed, with the A subunit NTD forming a local dimer with the B subunit NTD and the C subunit NTD forming a local dimer with the D subunit NTD, as has been previously described for AMPARs. Critically, while significant conformational heterogeneity exists within the GluA4 NTD, the trans-dimer interface between the B and D subunits appears to be preserved (Fig. 1b,c, Supplementary Fig. 3b), with the B and D subunits angled between 98.9-99.3° relative to one another, like what has been reported for native calcium permeable receptors unbound to Noelin41. All resolvable classes of the GluA4 NTD appear to swap local dimer partners below the NTD layer, with the A subunit switching from a local dimer with the B subunit at the level of the NTD to forming a local dimer with the D subunit at the level of the LBD, and the C subunit switching from a local dimer with the D subunit at the level of the NTD to a local dimer with the B subunit at the layer of the LBD (Fig. 1b). This contrasts with the NTD arrangement in GluA1 homotetramers where the NTD layer is ruptured with occasional non-domain-swapped GluA1 homotetramers42.

To measure the degree of conformational heterogeneity within the NTD layer, we refined maps of the LBD and TMD corresponding to the particle sets contributing to each NTD class (Supplementary Fig. 3a). Alignment of these maps resulted in composite maps in which differences between the classes in NTD conformation relative to the LBD and TMD layers could be observed, as was reported with GluA2/CNIH3 complexes45. Variable angles between NTD dimer pairs have been simulated44 and observed in crystal structures46, however, the NTD of GluA4 maintained an intact dimer-of-dimer architecture with the two local NTD dimer pairs angled between 42° and 46° relative to one another (class 9 and class 6, respectively; Supplementary Fig. 3c). The NTD remains intact in all resolvable classes but exhibits up to 14° of tilt relative to the pore axis. Furthermore, the distance between the bottom of the NTD and the top of the LBD layer was variable among the classes, exhibiting a 15 Å difference perpendicular along the pore axis (Supplementary Fig. 3c). Despite these differences in the NTD layer, differences in the overall conformation of the LBD and TMD layers were not observable. Taken together, these findings indicate that the NTD layer of GluA4 homotetramers is intact but exhibits an extraordinary degree of conformational heterogeneity.

Activation of GluA4
The degree of conformational heterogeneity that we observed in the NTD of activated GluA4 homotetramers suggests that the NTD may be able to act independently of the LBD and TMD layers to carry out functions distinct from channel gating. We therefore examined the activation mechanism of our purified GluA4 homomers, which were prepared in the presence of 1 mM Glu and 100 μM CTZ. Further processing of our consensus particle set produced a map of GluA4 homotetramers containing the LBD and TMD layers at an overall resolution of 3.31 Å, with local resolution below 3 Å in the channel pore (Supplementary Fig. 4). From this map, we observed all four LBD clamshell domains in the closed conformation with Glu bound in the LBD clamshell and CTZ present between the LBD domains (Fig. 2a, Supplementary Fig. 5). As previously seen with detergent-exchanged GluA2 complexes, lipid densities could be resolved around the TMD of the GluA4 assembly (Supplementary Fig. 6)45,47,48. These lipids appear distinct from the previously reported cholesterol densities associated with AMPAR/TARP complexes45,47,48 although resolution limitations preclude assignment of lipid identities with high confidence.

To confirm that the pore is open in our GluA4 active state, we measured the radius of the ion channel along its z axis. The GluA4 active state measured 2 Å at the most constricted point of the upper channel gate (A643 in GluA4, A642 in WT GluA2), indicating an open channel (Fig. 2b). Together, these data indicate that the conformational heterogeneity in the GluA4 NTD does not meaningfully limit the ability of the GluA4 LBD to activate the channel.

Gating in AMPA-type glutamate receptors is achieved by translating the motion generated by LBD clamshell closure through the M3 linkers and resulting in deformation of the M3 helices, sufficient to open the bundle crossing at the upper channel gate. These deformations have been referred to as ‘kinks’ in the M3 helix. As in other AMPAR structures, gating is dominated by deformations of the M3 helices in the B and D subunits within the “ISSYTANLAAF” motif that couples the top of the M3 to the lower D2 lobe of the LBD (Fig. 2c). This kink is defined by a loss of helical character which differs between the B and D subunit M3 helices. The B subunit M3 helix adopts a formation reminiscent of the ‘kinking’ found in other active state AMPAR structures, with a prominent bend in the helix beginning at T639 which returns to a more standard helical conformation at L642 before finally unwinding at F645. In contrast, at the D subunit, the helix appears to simply unwind beginning at T639. M3 helical character ends in both the A and C helix beginning at N641, with a short restoration of helical character between L642 and A644 (Fig. 2d). Kinking in the A and C helices has been previously reported for GluA2 homotetramers activated at physiological temperatures49, but was not detected in a recent activated state of GluA4 homotetramers50.

Conformational plasticity in the GluA4 LBD
Our structure of the GluA4/TARPγ2 complex revealed the activated state. However, close examination of single channel currents from purified GluA4/TARPγ2 complexes exposed to the same saturating concentrations of Glu and CTZ indicated the presence of multiple distinct levels of conductance through GluA4/TARPγ2 channels (Figs. 1a, 3a). We made recordings of single GluA4/TARPγ2 channels in planar lipid bilayers in both the absence (Fig. 3a) and presence of physiological concentrations of Ca2+ (1.5 mM, Fig. 3b). Channels opened readily in the presence of 1 mM Glu and 100 μM CTZ, often for tens of seconds at a time (Fig. 3a, top trace). Multiple channel conductances were reached directly from the closed state (Fig. 3a, bottom trace) and changes in channel conductance observable within open periods in the presence of Ca2+ (Fig. 3b). Analysis of channel openings revealed a distribution of conductances consistent with previous reports of four discreet conductance states for single GluA4/TARPγ2 channels37. Frequency histograms charting the amplitude of channel openings were plotted for channels recorded in the absence (Fig. 3c) and presence of 1.5 mM Ca2+ (Fig. 3d) and both histograms could be fit with four Gaussian curves, each corresponding to a different magnitude of channel opening. GluA4/TARPγ2 channels recorded in the absence of calcium exhibited conductance states of 13, 29, 38 and 54 pS, representing O1, O2, O3, and O4, respectively (respective standard errors of curve centers: 0.0323, 0.00856, 0.02252, 0.06306). Channels recorded in the presence of 1.5 mM Ca2+ exhibited channel conductances of 11, 24, 36, and 50 pS, corresponding to O1, O2, O3, and O4, respectively (respective standard errors of curve center: 0.00389, 0.00608, 0.0071, 0.02483). Comparing the mean conductances of all channel openings between channels recorded in the presence or absence of Ca2+ revealed a mean conductance of 37.15 pS in the absence of calcium compared to 25.67 pS in the presence of 1.5 mM Ca2+ (Fig. 3e). These data suggest that while GluA4/TARPγ2 homotetramers are permeable to Ca2+, that calcium stabilization in the open channel may reduce the overall conductance of these receptors in the presence of Ca2+, possibly through interaction with a recently reported binding site for Ca2+ in the upper channel gate of AMPARs, or through an inhibitory mechanism8.

Given the presence of multiple conductance states in our single-channel datasets (Fig. 3f), we wondered if this may also be true for our cryo-EM dataset, which was collected in the absence of Ca2+. The GluA4/TARPγ2 local resolution map indicates the LBD layer is conformationally heterogeneous compared to the pore-forming TMD (Supplementary Fig. 4). We used 3D Variability Analysis (3DVA) to identify components of variability within our consensus particle set (Supplementary Fig. 7) that may explain how variable conductances arise within a population of active state receptors. 3DVA revealed one prominent variability component which appeared to show conformational heterogeneity manifesting as a ‘rocking’ or ‘twisting’ of the LBD layer relative to the TMD layer51 (Supplementary Movie 1).

We separated the particles in our consensus dataset along an axis represented by this variability component (Variability Component 0) which resulted in a Gaussian distribution of particles along the axis. We then binned the particles into five individual groups along the Gaussian distribution of this variability component and generated five independent maps representing different ‘substates’ of the active state receptor (Fig. 3g, Table 1).

These substate maps varied in the number of contributing particles and their overall resolution but were each sufficiently well resolved to build five individual models each representing a different LBD/TMD relationship (Supplementary Figs. 8–9). These maps and resulting models revealed that our activated state was an average of a heterogeneous population of receptors along this variability axis, which represented variable positioning of the LBD to the TMD.

GluA4 substates exhibit variable channel openings
Since opening of the AMPAR ion channel requires LBD closure to pull open the channel gate, we speculated that variable LBD positions might alter the efficiency of this process and therefore the magnitude of pore opening. We measured the pore radius of each of our solved substates using HOLE52 and found two sites of variable channel opening between the substates (Fig. 3h). The most pronounced site of variability in the upper channel gate was at A643 which showed 2 different substates varying in constriction with a lower limit of 2.3 Å (Substate 2) and an upper limit of 2.9 Å (Substate 1, Fig. 3h). Notably, both of these measurements revealed a larger radius that was measured from the consensus structure (2.0 Å) The channel was further variable between the substates at T639 (2.7–3.1 Å), which is vital for Ca2+ binding8, as well as at the top of the selectivity filter, Q608 which ranged from 1.6 Å to 2.5 Å in channel radius.

How are these different substates achieved? Alignment of the TMD layer of the substates revealed overall similar TMD conformations (RMSD=0.8-1.3 Å) while the LBD layer exhibited more heterogeneity (RMSD=1.0-3.2 Å; Supplementary Fig. 10a). Notably, each of the substates showed all four LBD clamshells in the closed conformation around a glutamate molecule and CTZ was present between each LBD local dimer pair (Supplementary Fig. 10b-e), indicating that the substates did not vary in their ability to bind to the agonist Glu or the positive allosteric modulator CTZ. Close inspection of the substate maps revealed different conformations of the M3 helix critical for channel gating (Supplementary Fig. 9). Interpolation between the substates revealed a vector of heterogeneity that exhibited variable rotation of the LBD layer with respect to the TMD (Supplementary Movie 2). This rotation about the pore axis proceeded clockwise from Substate 1 to Substate 5, with 7° of overall rotation of the LBD local dimers (Fig. 4a). In addition, all four LBDs rotated downwards away from the pore axis an additional 18° in Substate 5 compared to Substate 1 (Fig. 4b). The entire LBD layer rotates as a rigid body 5° perpendicular to the pore axis (Fig. 4c).

Given that transmission of motion in the LBD to the M3 linkers and TMD underlies channel gating during activation, we investigated how these motions were transferred from the LBD to the TMD layer. Examination of the B and D helices of each substate revealed that the site of helical disruption changed between substates (Fig. 4d). In Substates 1 and 3, the B and D helices both lost helical character at T639, with helix D exhibiting more of an unwinding N-terminal of N641 rather than a kink. In contrast, in Substate 2, the B helix kinked at S637 with the D helix kinking at T639. In Substate 4, the B helix exhibits a compound kink at S637 and the D helix exhibits a kink at Y648 (Fig. 4d). Substate 5 shares the compound B helix kink of Substate 4 but unwinds the kink begins at Y638. The D subunit in substate 5 kinks at Y638 before unwinding completely at N641.

In the A and C helices, the location of the gating hinge progressed deeper through the substates. Substates 1, 2 and 3 progressively hinge deeper within the pore, starting with T647 in Substate 1 and progressing to A644 in Substate 3. In Substates 4 and 5, the hinge in the C helix kinks at A640, even while the A helices kink at L646 and F645 (Fig. 4e). Interestingly, the most kinked states in the A and C helices are not the most open at the upper channel gate, indicating a potential complex relationship between hinging in the A and C helices and conductance (Fig. 3h).

Among all substates and the consensus, we have identified eight residues in which a loss of helical character in the M3 helices facilitates channel gating (S637, Y638, T639, A640, N641, A644, F645, and L646). Of these only A640 (A618 in GluA2, the “A-hinge”), S637 (the “S-hinge”) and A644 (the “Lurcher Hinge”)49,53 have been previously reported. When including all known hinging residues appears that almost every residue in the 637-SYTANLAAF-645 motif common to ionotropic glutamate receptors is competent for forming a gating hinge, explaining why this motif is so thoroughly conserved among ionotropic glutamate receptors1 and why mutations in this motif produce pathological outcomes in human patients. Indeed, mutations at N641, T639, and A644 in GluA4 have recently been identified in human patients experiencing intellectual disability and epilepsy17. Collectively, these data indicate that the magnitude of channel opening in GluA4 AMPARs is at least partially controlled by the magnitude and location of the helical kinks in A and C subunits, and that this can be tuned by the conformational plasticity in the LBD layer.

Methods
Construct design
A fusion construct consisting of the coding sequence of GluA4 fused to TARPγ2 was generated based on previous strategies for generating AMPAR expression constructs and a similar construct used for studying GluA4 physiology (Supplementary Fig. 1)37,42,47,48,53,70. This construct is referred to as GluA4-γ2 and was prepared as follows. Rat GluA4 was truncated after the fourth transmembrane helix, and a single Glycine-Threonine (GT) linker was inserted between the GluA4 truncation and the N-terminus of the human TARPγ2 coding sequence. The TARPγ2 coding sequence was itself truncated after the fourth transmembrane helix (TM4), followed by a Threonine-Glycine-Glycine (TGG) linker, a Thrombin cleavage site, a short polyalanine stretch and then the coding sequence for eGFP. Finally, a StrepTag II was appended to the end of the coding sequence prior to the stop codon to facilitate protein purification.

Protein expression and purification
Bacmid for the expression of GluA4-γ2 was prepared as previously described47,53,70,71. P1 baculovirus was generated by transfecting ExpiSf9 cells (Gibco, A35243) maintained at 27°C with polyethyleneimine (PEI, molecular weight 40,000; PolyScience, 24765). After 5 days P1 baculovirus was harvested and used to induce expression in mammalian cells. Media containing P1 virus was added to cultures of Expi293F GnTI- cells (Gibco, A39240) grown to a density of 2.5 × 10^6 cells/mL in Expi293 medium (Gibco, A14135101) at a ratio of 1:10 P1 virus to Expi293F culture volume. Infected Expi293F cells were incubated at 37°C for 12–18 h before bringing the culture medium up to 10 mM sodium butyrate (Sigma, 303410) and 2 μM ZK20075 (Tocris, 2345) and incubating the culture at 30°C in 5% CO2. 72 h after transduction, the cells were harvested by low-speed centrifugation (5000 x g, 20 min at 4°C), washed with PBS (pH 7.4) with protease inhibitors (0.8 μM aprotinin, 2 μg/ml leupeptin, 2 μm pepstatin A and 1 mM phenylmethylsulfonyl fluoride) and then centrifuged a second time (4800 x g, 10 min at 4°C). Supernatant from this spin was discarded and the cell pellets were stored at −80°C until purification. On the day of purification, pellets were thawed by rotating the pellet in chilled lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0) with protease inhibitors added. After thawing, cells were lysed in an ice bath with a blunt probe sonicator (three cycles of 1 s on, 1 s off for 1 min, 20 W power), and the sonicated lysate was gently centrifuged (4800 x g, 20 min at 4°C) to clear large cellular debris. The supernatant was ultracentrifuged (125,000 x g, 45 min at 4°C) to pellet membranes. Solubilization buffer (150 mM NaCl, 20 mM Tris pH 8.0, 1% n-dodecyl-β-D-maltopyranoside (DDM; Anatrace D310) and 0.2% Porcine Brain Total Lipid Extract (Avanti, 131101P)) was added to the pelleted membranes and membranes were solubilized for 2 h at 4°C in a cold room under constant agitation. To prepare the brain total lipid extract for the solubilization buffer, 1 g of DDM and 200 mg of brain total lipid extract were added to a 50 mL conical tube and brought to a 50 mL volume with tris buffer (150 mM NaCl and 20 mM Tris pH 8.0) and incubated rotating at 4 C overnight. After overnight incubation, some of the brain total lipid extract remained insoluble. Solubilized membranes were then ultracentrifuged (125,000 x g, 45 min at 4°C) to remove insoluble material and the supernatant was applied to a pre-equilibrated volume of Strep-tactin XT 4Flow resin (IBA, 2-5010) overnight, rotating at 4°C. The next day, the resin was collected by gravity flow and washed with 20 volumes of glycol-diosgenin (GDN) buffer (150 mM NaCl, 20 mM Tris pH 8.0 and 0.01% GDN (Anatrace, GDN101)). Bound protein was eluted with 50 mM D-biotin in GDN buffer and collected directly into a centrifugal concentrator and concentrated into a 500 μl volume at 4°C. The concentrated protein was then treated with thrombin at a 1:200 w/w ratio for 1 h at 22°C to cleave off eGFP and Strep Tag II. The cleavage reaction was separated over a Superose 6 increase 10/300 column (Cytiva, 29091596) using an AKTA fast protein liquid chromatograph in GDN buffer. Two peaks were observed on the chromatogram corresponding to a higher and lower molecular weight species for GluA4. Peak fractions corresponding to the lower molecular weight species were collected and concentrated in a centrifugal concentrator to 1.49 mg/ml. Porcine Brain Polar Lipid Extract (Avanti, 141101P) was tested for solubilizing receptors but was not used for the final purification (Supplementary Fig. 1b).

Cryo-EM sample preparation and data collection
Purified GluA4/TARPγ2 was treated with 100 μM cyclothiazide (CTZ, Tocris, 07-131-0) and ultracentrifuged (125,000 x g, 45 min at 4°C) to remove precipitated protein prior to grid preparation. Au-Flat 1.2 μm/1.3 μm 300 mesh gold grids (MiTeGen, M-CEM-AUFT313) were plasma treated in a Pelco Easy Glow (25 mA, 120 s glow time and 10 s hold time; Ted Pella, 91000). The GluA4/TARPγ2 complexed with CTZ was spiked with glutamate (pH 7.4) immediately prior to the preparation of grids. To prepare grids, 3 μl of sample was applied to glow-discharged grids in an FEI Vitrobot Mark IV (Thermo Fisher Scientific; wait time 30 s; blot force 5, blot time 4 s) set to 8°C and 100% humidity and plunge frozen in liquid ethane. Grids were imaged with a 300-kV Titan Krios 3i microscope equipped with fringe-free imaging, a Falcon 4i camera and a Selectris energy filter set to a 10-eV slit width. Micrographs were collected with a dose rate of 3.83 e- per pixel per s and a total dose of 40.00 e- per Å2. We collected 19,054 micrographs with a pixel size of 0.93 Å per pixel. Automated collection was achieved with EPU software from Thermo Fisher Scientific.

Image processing
Cryosparc72 was used for all aspects of image processing (refer to Supplementary Figs. 2 and 7 for details). The reconstruction quality was tested for anisotropic contribution to the Fourier shell correlation (FSC) with 3DFSC.

Model building, refinement, and structural analysis
Molecular modeling was performed in ChimeraX73 and Coot74. Refinement was performed with a combination of ISOLDE75 and PHENIX76. These software packages were accessed through the SBgrid consortium77. As a starting model, four copies of the LBD and TMD layers of AlphaFold2 prediction for GluA4 were each rigid-body fit into the consensus model and then each protomer LBD and TMD layer was joined. Four copies of the previously solved model of TARPγ2 were then rigid body fit into each corresponding density to create a single consensus model. ISOLDE was then used to adjust the position of all amino acids simultaneously before inspecting each amino acid fit to the map and merging Glu and CTZ into the model in Coot. Phenix was used to refine the final model. MolProbity78 was used to assess model quality. Visualization, figure generation and domain measurements were performed in ChimeraX. HOLE was used to measure the radius of the ion channel pore along the z axis52.

Single channel recording
Measurement of GluA4/TARPγ2 single channels was recorded in reconstituted lipid bilayers using Meca 4 Recording chips (100 μm cavity; Nanion, 132002) in an Orbit Mini device (Nanion) with the Elements DataReader-4 software (Elements S.R.L./Nanion) as previously reported41. Briefly, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPHPC, Fisher Scientific, NC0652354) was solubilized at 10 mg/ml in decane (Sigma-Aldrich, D901-100ML) and used as lipid stock. A thin lipid bilayer was painted over each recording cavity on a Meca 4 chip equilibrated in recording solution (150 mM KCl, 20 mM HEPES pH 7.5, 1 mM Glutamate, 100 μM CTZ, with or without 1.5 mM Ca2+), by gently dispensing lipid-covered air bubbles over the cavities from a 10-µl tip previously dipped in the lipid stock. Purified GluA4/TARPγ2 sample was diluted to ~100 ng/ml in the recording solution, and 4 µl of diluted protein was added directly onto top of each painted cavity. The chip was washed with fresh recording solution several times to remove residual proteins after each round of the recordings, and fresh lipid bilayers were repainted prior to the following data collection. The capacitances of painted bilayers were kept between 10−25 pF. The recordings were conducted at –60 mV holding potential with a 200 pA applied current rate. The data were sampled at 20 kHz. Analysis of recordings was performed in Clampfit 11 (Axon Devices) following the application of an 8-pole Bessel filter to smooth the recordings. Analysis was restricted to periods in which at least one channel opening was clearly visible. A frequency histogram of channel openings was constructed in Origin and was fit with using the nonlinear curve fit feature as a Gaussian with four peaks.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I thank the authors for their efforts in addressing my questions and revising the manuscript. They have responded thoroughly by incorporating updated results and adding relevant explanations, primarily in the Discussion section. The new data are more robust than in the original submission, and the expanded discussion provides clearer context and more nuanced interpretations. Overall, I am satisfied with how my concerns have been addressed.\nHowever, one issue remains regarding the Ramachandran outliers. Although the authors mention in their response that this has been revised, the final model table and the PDB validation report still contain the outlier numbers. I double-checked two models—the consensus structure and substate 5—and both still show 3–5 outliers, which was not addressed in the rebuttal. This point appears to have been overlooked.\nAdditionally, one of my earlier questions regarding the five substates remains unanswered. Do the authors believe that these five substates represent all major conformational intermediates? Could larger cryo-EM datasets reveal additional states that are currently missing?", "reviewer_2": "AMPA receptors made of GluA4 subunit are calcium permeable and expressed in PV positive interneurons and cerebellum, where they express distinct gating characteristics and synaptic plasticity. The gating mechanism of this important AMPA receptor subtype is incompletely understood. Hale et al. investigates this problem by reporting the open gate cryo-EM structures of GluA4 flip isoform with TARPgamme2 auxiliary subunit. The construct design follows their previously established GluA2-TARPgamma2 fusion. The activated open gate conformations were captured by applying CTZ and glutamate. The complex was extracted from the cells using DDM/total brain lipid and then exchanged to GDN to improve stability for cryo-EM analysis, an optimal condition that is different from other AMPAR subunits.\nThe work has been revised thoroughly in response to previous reviews. The cryo-EM maps and models are improved. The new data is provided on the single channel physiology in reconstituted lipid bilayer of the GluA4-TARPgamma2, showing four conductance states with or without physiological concentration of Ca2+. They demonstrate Ca2+ reduces the conductance of all four sub conductance states, and at the same time alter the occupancy among those states. They suggest, based on high occupancy of the open channels in O3 and O4, that the cryo-EM structures captured must be in those sub conductance states.\nThe structures of the NTDs are carefully analyzed. The NTDs form dimer-of-dimer assembly but exhibit extensive structural heterogeneity in the way the NTD layer is positioned relative to the rest of the complex. Despite these variety, they appear to have little impact on the organization of the LBDs and the TMD.\nThe structural characterization of the M3 gate kink is clearly described.\nOne of the significant findings is that the kink conformation relates to the way four activated LBDs are spatially arranged. The 5 structural substates exceed the number of sub conductance states (O1 to O4) measured by channel recordings, which casts interesting idea that more than two conformations could map to the same sub conductance state.\nOverall, the work presents new and significant information about the gating mechanism of GluA4 TARPgamma2 AMPA receptor complex and how sub conductance states relates to receptor conformation. I have only several minor issues as listed below.\nMinore comments:\nThe reference should be provided for the statement in line 137.\nCould the asymmetric kink of the open gate relate in any way to the asymmetric positioning of the NTD layer? Where the asymmetry comes from is an interesting question.\nStructural variation of the LBD layers in the open GluA2 TARPgamma2 complex has been reported previously in Nakagawa et al 2024 (ref 8). They show three possible configurations of the LBD gating ring in the Extended Fig 5A-E. This point could be introduced and discussed.\nLine 270 and 273: The reference to the figure should be Fig.3H.\nSome discussion on the structural difference between the authors and the recent GluA4 TARPgamma2 open gate structure reported\nfrom the Herguedas lab might be considered. Could it be the Ca2+?"}, "author_response": "We are excited that the reviewers agree that our manuscript has been improved during the revision process.\nWe address their points below.\nReviewer #1 (Remarks to the Author):\nI thank the authors for their efforts in addressing my questions and revising the manuscript. They have responded\nthoroughly by incorporating updated results and adding relevant explanations, primarily in the Discussion section.\nThe new data are more robust than in the original submission, and the expanded discussion provides clearer context\nand more nuanced interpretations. Overall, I am satisfied with how my concerns have been addressed.\nHowever, one issue remains regarding the Ramachandran outliers. Although the authors mention in their response\nthat this has been revised, the final model table and the PDB validation report still contain the outlier numbers. I\ndouble-checked two models—the consensus structure and substate 5—and both still show 3–5 outliers, which was\nnot addressed in the rebuttal. This point appears to have been overlooked.\nAdditionally, one of my earlier questions regarding the five substates remains unanswered. Do the authors believe\nthat these five substates represent all major conformational intermediates? Could larger cryo-EM datasets reveal\nadditional states that are currently missing?\nResponse: Thank you for the comments, and for the enthusiasm toward our revision.\nRegarding the model outliers, these outliers reflect the quality of our cryo-EM data and the fact that\nexperimental modeling is not perfect. These outliers are minimal compared to similar studies (e.g.,\nGangwar...Sobolevsky NComms 2024; Kang…Furukawa Nature 2025; Mondal…Twomey Nature 2025;\nWang…Twomey Nature 2025), and having perfect geometry in any cryo-EM based study would not accurately\nreflect local resolution differences in cryo-EM data nor experimental variation (e.g., Richardson…Richardson\nIn our experimental conditions, these substates represent the major conformational intermediates. We agree\nthat collecting additional data to populate rarer states would enable reconstruction. However, it is impossible\nto project the dataset size that this would require. We edited the text accordingly to make these points more\nclear.\nReviewer #2 (Remarks to the Author):\nAMPA receptors made of GluA4 subunit are calcium permeable and expressed in PV positive interneurons and\ncerebellum, where they express distinct gating characteristics and synaptic plasticity. The gating mechanism of this\nimportant AMPA receptor subtype is incompletely understood. Hale et al. investigates this problem by reporting the\nopen gate cryo-EM structures of GluA4 flip isoform with TARPgamme2 auxiliary subunit. The construct design follows\ntheir previously established GluA2-TARPgamma2 fusion. The activated open gate conformations were captured by\napplying CTZ and glutamate. The complex was extracted from the cells using DDM/total brain lipid and then\nexchanged to GDN to improve stability for cryo-EM analysis, an optimal condition that is different from other AMPAR\nsubunits.\nThe work has been revised thoroughly in response to previous reviews. The cryo-EM maps and models are\nimproved. The new data is provided on the single channel physiology in reconstituted lipid bilayer of the GluA4-\nTARPgamma2, showing four conductance states with or without physiological concentration of Ca2+. They\ndemonstrate Ca2+ reduces the conductance of all four sub conductance states, and at the same time alter the\noccupancy among those states. They suggest, based on high occupancy of the open channels in O3 and O4, that the\ncryo-EM structures captured must be in those sub conductance states.\nThe structures of the NTDs are carefully analyzed. The NTDs form dimer-of-dimer assembly but exhibit extensive\nstructural heterogeneity in the way the NTD layer is positioned relative to the rest of the complex. Despite these\nvariety, they appear to have little impact on the organization of the LBDs and the TMD.\nThe structural characterization of the M3 gate kink is clearly described.\nOne of the significant findings is that the kink conformation relates to the way four activated LBDs are spatially\narranged. The 5 structural substates exceed the number of sub conductance states (O1 to O4) measured by channel\nrecordings, which casts interesting idea that more than two conformations could map to the same sub conductance\nstate.\nOverall, the work presents new and significant information about the gating mechanism of GluA4 TARPgamma2\nAMPA receptor complex and how sub conductance states relates to receptor conformation. I have only several minor\nissues as listed below.\nMinore comments:\nThe reference should be provided for the statement in line 137.\nResponse: This was added to the text.\nCould the asymmetric kink of the open gate relate in any way to the asymmetric positioning of the NTD layer? Where\nthe asymmetry comes from is an interesting question.\nResponse: Thank you for this point, we add this possibility to the text, and could hint how trans-synaptic\nfactors alter function.\nStructural variation of the LBD layers in the open GluA2 TARPgamma2 complex has been reported previously in\nNakagawa et al 2024 (ref 8). They show three possible configurations of the LBD gating ring in the Extended Fig 5A-\nE. This point could be introduced and discussed.\nResponse: We added this point to the discussion.\nLine 270 and 273: The reference to the figure should be Fig.3H.\nResponse: Thank you for catching this, we corrected this in the text.\nSome discussion on the structural difference between the authors and the recent GluA4 TARPgamma2 open gate\nstructure reported\nfrom the Herguedas lab might be considered. Could it be the Ca2+?\nResponse: This is an interesting possibility – thank you! We added this to the discussion."}]

Decision letter