Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers

Abstract

The assembly of AMPA-type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo- and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane-distal AMPAR N-terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit-specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure-guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This 'flexibility' of the NTD not only explains why heteromers predominate but also how GluA2-lacking, Ca(2+)-permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo- and heteromeric AMPAR tetramers.

Figure 1

GluA1–4 NTDs exhibit markedly different assembly behaviours. (A) Homomeric NTD assembly properties. Sedimentation coefficient (S) distributions were obtained for FAM-labelled GluA1–4 NTDs at 50 nM protein concentration. Labelled species are denoted with an asterisk (^*). Continuous distribution c(S) models revealed a spectrum of homomeric affinities, from low (top) to high (bottom). Two species at ∼2.6S (monomer) and 3.7S (dimer) could be detected for all NTDs. GluA1^* (green trace) showed incomplete homodimerization, as indicated by presence of the monomer peak at 2.6S. In contrast, GluA2^* (red trace) and GluA4^* (blue trace) were mostly dimeric, whereas GluA3^* was mostly monomeric (grey trace). (B) Heteromeric NTD assembly properties. c(S) distributions are shown for FAM-labelled GluA3^* (top) or GluA1^* (bottom) at monomeric concentrations mixed with non-labelled GluA2 NTD. Adding non-labelled GluA2 was sufficient to shift the labelled monomeric species to a dimeric peak, indicating the presence of heterodimers. (C) Summary of the distinct assembly properties of GluA1–4 NTDs. In GluA1, GluA4 (left pathway), and GluA2 (top), heteromerization is favoured but must compete with possible homomerization, leading to preferential heterodimers; whereas in GluA3 (right pathway), homomers are disfavoured and therefore the NTD exhibits obligatory heteromerization. See also Supplementary Figures S1 and S2.

Figure 2

Primary, secondary, and tertiary structure characteristics of the GluA2 NTD dimer interface. (A) Position-specific patterns of conservation in the GluA1 and GluA2 upper lobe (UL) interface. GluA1-specific, GluA2-specific, and consensus residues, generated from partitioning an alignment of 34 GluA1 and 33 GluA2 sequences, are indicated for positions in helices B and C, the major contributors of the UL interface. The top loop (not shown) is fully conserved between GluA1 and GluA2. N54 and T78 in GluA2 were identified as potential key determinants for assembly specificity, because they are located at the interface and are systematically mutated between GluA1 and GluA2. (B) The molecular surface of the GluA2 NTD is shown with UL and LL interfaces coloured dark and light blue, respectively, with the contribution of the variable interface residues shown in yellow. (C) Secondary structure contributions to the dimer interface. The NTD is oriented as in B, highlighting helices B and C in the UL-dimer and helix E and sheet 7 in the LL-dimer interfaces, respectively. Positions of N54 and T78 are in yellow, and important LL-interface contacts (L137, Q141, L144) on helix E are shown in orange. (D) Model of a potential heteromeric GluA1/GluA2 interface. A homology model of GluA1, associated with the crystal structure of GluA2 (3HSY) is shown, zoomed in on the T78 (A2)–M78 (A1) interaction in yellow.

Figure 3

Mutation in the NTD perturbs assembly of the full-length receptor. (A) Blue native PAGE (BN-PAGE) analysis of NTD interface mutants shows differential migration patterns. HEK293T cell suspensions of GluA2 flop (R/G unedited, Q/R edited) wild type (WT) and mutants (indicated on the top) were separated on 4–12% BN-PAGE and visualized by western blotting. Monomeric (M), dimeric (D), and tetrameric (T) assembly intermediates are denoted. Note the different phenotypes of N54A and T78A, as well as the intermediate phenotype of the N54A/T78A double mutant. (B) Mild SDS treatment before BN-PAGE dissociates tetramers and reveals that N54A (stabilizing) and T78A (destabilizing) have a bidirectional effect on tetramer and dimer assembly, relative to the wild type. The gel shown on right was scanned and bands computed as intensity peaks using ImageJ software (NIH).

Figure 4

Mutation of the upper lobe (UL) interface results in bidirectional changes in heteromeric assembly. (A) Representative voltage clamp recordings of GluA2 NTD mutants in outside-out patches when co-transfected into HEK293T cells at a fixed, limiting ratio for heteromeric assembly with GluA1. Only currents for −60, 0, and +40 mV are shown to illustrate the differences in outward current for a series of key mutants. Currents are normalized to the absolute value at −60 mV. (B) Averaged conductance–voltage (G–V) plots for all patches of mutants described in A. Chord conductance (G) is normalized to the absolute value at −60 mV. Number of patches is shown in brackets. The dashed line denotes the G–V curve for homomeric GluA1 (n=4). Error bars represent s.e.m. and are only shown for the positive deviation. (C) Mutation of the conserved hydrophobic cluster in the UL interface in the GluA2 NTD generally disfavours heteromeric assembly with GluA1. I–V relationships were quantified by determining the slope conductance (g) at +10 and −40 mV and expressing these as a ratio, g₊₁₀/g₋₄₀, or rectification index (RI). The geometric mean of the RI is plotted on a logarithmically scaled axis, with increasing RI indicating greater heteromerization. Error bars show the back-transformed limits of the s.e.m. of the log_e RI data. Number of patches is given at the base of each column. The dashed line denotes the RI value for WT GluA2. FF=F50A/F82A, FFL=F50A/F82A/L310A, FL=F82A/L310A. (D) Mutation of polar residues in the UL generally favour heteromeric assembly. Rectification indices of the indicated mutants are represented as in Figure 3D. Note that double mutation of N54 and T78 shows no additive effect and actually decreases heteromerization when mutated to GluA1 residues. The dashed line denotes the RI for GluA2 WT. ^**P<0.01 (one-way ANOVA with Dunnett multiple comparison: mutants versus wild type). (E) Molecular surface of the GluA2 dimerization interface is shown, with residues targeted for mutagenesis shown as either facilitating (green) or inhibiting (blue) heteromerization. See also Supplementary Figure S3.

Figure 5

Mutation of variable residues in the GluA1 upper lobe (UL) show increased heteromerization competence. (A) Voltage clamp recordings of GluA1 NTD mutants in outside-out patches when co-transfected into HEK293T cells with GluA2. Only currents for −60, 0, and +40 mV are shown. Currents are normalized to the absolute value at −60 mV. (B) Averaged conductance–voltage (G–V) plots for all patches of mutants described in A. Chord conductance (G) is normalized to the absolute value at −60 mV. Number of patches is shown in brackets. The dashed line denotes the G–V curve for homomeric GluA1 (n=4). Error bars represent s.e.m. and are only shown for the positive deviation. (C) Mutation of key polar interface residues in the GluA1 NTD UL favours heteromeric assembly. Rectification indices of the indicated mutants plotted as in Figure 3D. The dashed line denotes the RI value for WT GluA1. ^*P<0.05; ^**P<0.01 (one-way ANOVA with Dunnett multiple comparison: mutants versus Wild type).

Figure 6

Structural basis for balanced assembly. (A) Top: Crystal structure of the GluA2-N54A NTD shows subtle alterations in the dimeric packing. Structural alignment of the N54A (green) and the WT (grey) crystal structures are shown. As expected, disrupting the hydrogen bond between N54 and the L310 main-chain carbonyl resulted in a different top loop conformation, but the L310 side chain was not shifted from its interface location. 2F_o−F_c omit map of A54 contoured to 1.0σ is shown for the relevant residue (blue) and the neighbouring residues (grey) in N54A. Bottom: In the WT context (PDB 3HSY), N54 makes a cross-dimer polar contact only on one side of the dimer interface (circled in blue). The result is unsatisfied hydrogen bonding potential at the dimer interface (circled in red), a destabilizing component of the WT interface. (B) Model of the thermodynamic effect of NTD mutations and their effects on whole-channel assembly. Whereas WT GluA2 NTD (top) is capable of balancing homo- and heteromerization at the level of the NTD and the whole receptor, N54A and T78A (bottom), which have opposite effects on NTD dimer stability, preferentially heteromerize the whole channel. Free energy changes of dimer dissocation (ΔΔG) in kcal mol⁻¹ are denoted. (C) Two interconnected steps dictate dimer assembly: homodimer dissociation (step I) and optimal homo- and heterodimer association (step II). Hotspots (stars) in the GluA2 NTD (red) upper lobe interface affect both these processes. Whereas the T78A mutation facilitates homomer dissociation, the N54A mutation stabilizes both homomerization and heteromerization (K_ds of dimer dissociation measured by AU-FDS are shown below the relevant assemblies). As heterodimerization requires both homodimer dissociation and re-association of heterodimers, the functional outcome for both mutations in the context of the full receptor is, therefore, greater heteromerization. The GluA1 NTD is shown in green for reference.

Figure 7

Differential affinities drive specific assembly. Summary of the experimentally derived association affinities. Note that the association affinities of NTDs span four orders of magnitude, from very tight K_ds below 2 nM (bottom) for assembly driven by GluA2, to intermediate K_ds in the range of 30–100 nM (top left) for assembly driven by GluA1 in the absence of GluA2, and to relatively loose, as in the case of Glua3 homomers (top right). Together with relative subunit concentrations, which differ in distinct neuronal populations (described in Supplementary Figure 6 and indicated by dashed outlines), these parameters will determine the assembly process.