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• W2113670233 abstract "Article11 February 2011free access Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers Maxim Rossmann Maxim Rossmann Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Madhav Sukumaran Madhav Sukumaran Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Andrew C Penn Andrew C Penn Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UKCurrent address: Centre National de la Recherche Scientifique UMR 5091, Cellular Physiology of the Synapse, Bordeaux, France Search for more papers by this author Dmitry B Veprintsev Dmitry B Veprintsev MRC Centre for Protein Engineering, Cambridge, UK Search for more papers by this author M Madan Babu M Madan Babu Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Ingo H Greger Corresponding Author Ingo H Greger Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Maxim Rossmann Maxim Rossmann Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Madhav Sukumaran Madhav Sukumaran Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Andrew C Penn Andrew C Penn Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UKCurrent address: Centre National de la Recherche Scientifique UMR 5091, Cellular Physiology of the Synapse, Bordeaux, France Search for more papers by this author Dmitry B Veprintsev Dmitry B Veprintsev MRC Centre for Protein Engineering, Cambridge, UK Search for more papers by this author M Madan Babu M Madan Babu Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Ingo H Greger Corresponding Author Ingo H Greger Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Author Information Maxim Rossmann1,‡, Madhav Sukumaran1,‡, Andrew C Penn1,‡, Dmitry B Veprintsev2, M Madan Babu3 and Ingo H Greger 1 1Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK 2MRC Centre for Protein Engineering, Cambridge, UK 3Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK ‡These authors contributed equally to this work *Corresponding author. Neurobiology Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK. Tel.: +44 122 340 2173; Fax: +44 122 340 2310; E-mail: [email protected] The EMBO Journal (2011)30:959-971https://doi.org/10.1038/emboj.2011.16 Current address: Centre National de la Recherche Scientifique UMR 5091, Cellular Physiology of the Synapse, Bordeaux, France PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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, Ca2+-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. Introduction Ion channels largely assemble into hetero-oligomers. Subunit heteromerization greatly expands the functional repertoire and is mediated by specific assembly domains (Schwappach, 2008). Ionotropic glutamate receptors (iGluRs), the main mediators of excitatory neurotransmission, are either obligatory (NMDA subtype) or preferential (AMPA, kainate types) heteromers (Traynelis et al, 2010). The subunit combination dictates fundamental signalling parameters such as gating kinetics and ion conductance, which shape synaptic physiology (Erreger et al, 2004). AMPA-type iGluRs (AMPARs), in addition to initiating excitatory signalling, adjust synaptic strength via dynamic postsynaptic trafficking (Malinow and Malenka, 2002; Greger and Esteban, 2007; Shepherd and Huganir, 2007), which underlies various forms of experience-dependent synaptic plasticity (Kessels and Malinow, 2009). These processes are mediated by mobilization and recruitment of distinct pools of AMPAR heteromers. AMPARs assemble from four subunits, GluA1–4, in various stoichiometries (Hollmann and Heinemann, 1994). Incorporation of GluA2 blocks Ca2+ flux through AMPARs; GluA2-containing receptors predominate throughout the brain (Isaac et al, 2007). The central role of AMPAR heteromer formation is well illustrated in the CA1 subfield of hippocampus. CA1 pyramidal neurons express comparable levels of GluA1 and GluA2 (but little GluA3 and no GluA4; Tsuzuki et al, 2001), which almost exclusively co-assemble (Lu et al, 2009). Pathological conditions can alter this balance, resulting in Ca2+-permeable, GluA2-lacking receptors and excitotoxicity (Kwak and Weiss, 2006; Liu and Zukin, 2007). GluA2-lacking receptors can also operate under specific physiological conditions (Cull-Candy et al, 2006), and have been detected in midbrain dopamine neurons, where enhanced signalling through these receptors is implicated in drug addiction (Carlezon and Nestler, 2002; Kauer and Malenka, 2007). How neurons control selective combinatorial assembly of subunits to generate this plethora of distinct compositional and functional phenotypes remains an open question. Furthermore, the composition of Ca2+-permeable (GluA2-lacking) AMPARs has not been established. AMPAR assembly is mediated by three domains engaging in distinct subunit interactions in the endoplasmic reticulum (ER): the N-terminal domain (NTD), the ligand-binding domain (LBD) and the membrane-embedded ion channel (Madden, 2002; Greger et al, 2007). The first assembly step is initiated by the NTD, which spans ∼50% of polypeptide sequence and primes subunit dimerization via an extensive bipartite interface (Clayton et al, 2009; Jin et al, 2009; Kumar et al, 2009). The NTD is therefore expected to provide a major assembly platform during iGluR biogenesis (Hansen et al, 2010). The second assembly step, association of dimers into tetramers, is modulated by Q/R editing in the channel pore (Greger et al, 2003). Relative to the NTD scaffold, contacts mediated by the LBD are weak but are regulated by RNA recoding events (Greger et al, 2006; Penn et al, 2008; Penn and Greger, 2009). The interplay between these domains and their roles in the assembly of functionally distinct receptor heteromers remain elusive (Sukumaran et al, 2011a). Here, we report that AMPAR heteromerization is driven by unexpectedly diverse, subunit-selective NTD interactions. A sensitive, fluorescence-based biophysical approach permitted direct quantification of homo- and heteromeric associations along the NTD axis. We find that GluA2 is dominantly incorporated into dimers at the level of the NTD and that assembly follows two distinct pathways: obligatory heterodimerization in case of GluA3 and preferential heterodimerization in GluA1 and GluA4. In the latter pathway, the non-constitutive, equilibrium-type assembly between homo- and heteromeric GluA1 NTDs potentially provides an explanation for the existence of GluA1 homomeric receptors, which have an enigmatic origin but have been detected under various conditions. Moreover, structural and functional mapping of the GluA1/2 NTD dimer interface identified evolutionarily variable hotspots mediating the balanced assembly function of the NTD, providing a mechanistic basis for the formation of Ca2+-permeable AMPARs. Therefore, a pivotal assembly function encoded in the NTD provides the organizing principle for the formation of diverse pools of AMPAR tetramers. Results Whereas tri-heteromeric NMDARs have been described (Hatton and Paoletti, 2005), an ongoing question for AMPARs is whether heteromers form initially at the level of the dimer or at the level of the tetramer via assembly of two different homodimers (Mansour et al, 2001; Gill and Madden, 2006). Solving this problem would hold the key to understanding the formation and organization of AMPAR heteromers. Because the NTD is believed to have a strategic role in subfamily-selective assembly (Leuschner and Hoch, 1999; Ayalon and Stern-Bach, 2001), we devised a sensitive, fluorescence-based ultracentrifugation assay to directly measure association of AMPAR NTD homo- and heterodimers. AMPAR subunit NTDs feature vastly different assembly properties GluA1–4 AMPAR subunit NTDs, purified to homogeneity, were subjected to analytical ultracentrifugation with fluorescence detection (AU-FDS) (Rajagopalan et al, 2008). This technique facilitates measurements of monomer/dimer equilibria in the nanomolar range (MacGregor et al, 2004), and provides a means to quantify both homo- and heteromeric affinities. NTDs were fluorescently labelled at the N-terminus with 5,6-carboxyfluorescein (FAM) and subjected to velocity sedimentation. As illustrated in Figure 1A, this approach revealed a surprising spectrum of subunit-selective oligomerization properties. At a concentration of 50 nM labelled protein, both mono- and dimeric species were detected for the GluA1 NTD. GluA2 predominantly sedimented as a dimer, whereas the GluA4 NTD showed an intermediate phenotype. In stark contrast, GluA3 was exclusively monomeric (Figure 1A). This sedimentation behaviour was protein-concentration dependent; for example, a greater proportion of GluA2 monomers were detected when lowering the input from 50 nM to 2 nM (Supplementary Figure S1A). By titrating the protein concentration, we were able to derive Kd values of dimer dissociation from multiple runs. We find that AMPAR NTDs exhibit a wide range of dimeric affinities, with a ∼1000-fold disparity between the GluA2 and GluA3 NTDs (1.8 versus 1200 nM, respectively). GluA1 and GluA4 fell between these extremes, with an estimated Kd of ∼100 and 10 nM, respectively (Table I). 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. Download figure Download PowerPoint Table 1. Association behaviour of NTDs assayed by analytical ultracentrifugation NTD protomers Kd (nM)a n measurements s.e.m. GluA1b 98 8 36 GluA2b 1.8 12 0.2 GluA3b 1200 12 500 GluA4b 10.2 9 1.1 GluA1b/GluA2 0.4 10 0.2 GluA3b/GluA1 38 9 6.0 GluA3b/GluA2 1.3 4 0.1 GluA4b/GluA2 3.3 9 0.15 GluA2-T78Ab 37.1 7 3.7 GluA2-N54Ab 0.5 4 0.2 Dissociation constant. FAM-labelled. Selective NTD affinities drive different assembly pathways The vast, ∼1000-fold range of homomeric affinities suggests different overall assembly behaviours for GluA1–4. As AU-FDS allowed us to test whether AMPAR subunits can heteromerize at the level of the NTD, and thus at the level of dimers, we asked how the drastic differences in homomeric affinities affect heteromer formation. Heteromers were assayed by holding the labelled species (denoted with an asterisk (*)) constant at monomeric concentrations and adding unlabelled NTD (Supplementary Figure S2). The presence of heteromers was indicated by a shift of the labelled monomeric species to a dimeric peak (Figure 1B). Kds were calculated by titration of unlabelled assembly partner. We derived a tight GluA1*/2 heteromeric Kd of ∼0.5 nM. Similarly, titration of GluA2 onto GluA3* and GluA4* revealed low Kds of 1.3 and 3.3 nM, respectively (Table I). Therefore, AMPAR NTDs heteromerize with greater affinity, when compared with their respective homomeric Kds (Table I). This result provides a ready explanation for the predominance of AMPAR heteromers, which was recently assessed in hippocampal pyramidal neurons where GluA1/2 receptors prevail (Lu et al, 2009). These data provide a roadmap for AMPAR heteromer assembly. First, they reveal two distinct AMPAR assembly pathways. Because of their relatively tight homomeric affinities, GluA1, GluA2 and GluA4 homomers are still likely to occur, but the existence of GluA3 homomers is unlikely in the presence of other subunits, especially GluA2 (Table I). Therefore, GluA1 and GluA4 preferentially heterodimerize with GluA2, whereas GluA2/3 dimers are obligatory (Figure 1C, bottom half). We note that GluA3 can form homomers in the absence of other assembly partners (e.g., Suzuki et al, 2008); hence, ‘obligatory’ heteromerization pertains only to GluA3 expressed in neurons. Interestingly, this result also illustrates an apparent assembly dominance of GluA2. Preferential formation of GluA2-containing AMPARs is a well-established phenomenon; our results suggest that this can be determined at early stages of receptor biogenesis via the NTD. In addition to the edited Q/R site prolonging GluA2 ER dwell time (Greger et al, 2002; Greger and Esteban, 2007; Sukumaran et al, 2011a), this finding could explain why AMPARs harbouring this functionally critical subunit widely dominate throughout neuronal populations (Isaac et al, 2007). In the preferential assembly pathway, GluA2 appears to have greater affinity for GluA1 than for GluA4 (Figure 1C, top half). Therefore, the equilibrium between homo- and heterodimers of these subunits with GluA2 will differ, which ultimately could also have consequences for the arrangement and number of GluA2 subunits in a tetramer (Washburn et al, 1997). Even though GluA2 has a dominant functional and assembly role, GluA2-lacking, Ca2+-permeable AMPARs have been detected and have a central physiological function (Cull-Candy et al, 2006; Kauer and Malenka, 2007). Their existence can be explained by the balanced assembly properties of the GluA1 (and GluA4) NTD (Figure 1A) and could be induced by increased GluA1 expression (Thiagarajan et al, 2005; Sutton et al, 2006; Aoto et al, 2008; see also Supplementary Figure S6). Hippocampal inter-neurons prominently express both GluA1 and 3 in the absence of GluA2 (Catania et al, 1998; Tsuzuki et al, 2001). In addition, a minor fraction of GluA1/3 co-precipitate from CA1/2 tissue, which increases in GluA2 knockout mice (Wenthold et al, 1996; Sans et al, 2003). The existence of pools of kinetically distinct, Ca2+-permeable AMPAR heteromers is therefore likely. Indeed, GluA1/3 NTDs produce a tight heteromeric Kd (∼38 nM; Table I) of greater affinity than their respective homomeric Kds. Similarly, GluA4/3 assemble in the low nanomolar range (data not shown). These data further suggest an obligatory heteromeric assembly mode for GluA3 in GluA2-lacking neurons and for the existence of functionally diverse Ca2+-permeable AMPAR populations. Importantly, the closely related GluK2 kainate receptor NTD does not shift GluA3* towards the dimer (data not shown); therefore, subfamily-specific assembly occurs at the level of the NTD. In summary, our results reveal that (1) MPAR heteromers form at the level of the dimer (which will affect the spatial arrangement of the channel tetramer), (2) subunit-selective assembly is driven by vastly distinct NTD associations, and (3) it results in preferential or obligatory AMPAR heteromers (Figure 1C). Properties of the NTD dimer interface The sedimentation data show that NTD heterodimers preferentially include GluA2 (Table I). This finding offers a solution for the long-standing question of how this key subunit is incorporated into the majority of AMPARs across neuronal populations; however, mechanistically, this poses an apparent paradox—how does the tight GluA2 homodimer permit selective heteromerization? Furthermore, our data also suggest that balancing between homo- and heteromerization via the NTD interface may explain the biogenesis of GluA1 homomers and possibly other populations of Ca2+-permeable AMPARs (Figure 1C). In vivo, neuronal populations tightly regulate synaptic recruitment of Ca2+-impermeable and Ca2+-permeable AMPARs; how these assembly modes are balanced is, therefore, critical for signalling dynamics (Toth and McBain, 1998; Cull-Candy et al, 2006; Liu and Zukin, 2007). To address these questions, we set out to pinpoint assembly determinants within the NTD interface. First, we mapped the evolutionary conservation within the GluA2 dimer interface, guided by our high-resolution GluA2 NTD structure (Supplementary Table I; Greger et al, 2009; Sukumaran et al, 2011b) and by position-specific comparative analysis of evolutionary conservation (Wuster et al, 2010). As conserved residues at interfaces are expected to maintain the affinity of subunit interactions (Landgraf et al, 2001), we first computed evolutionary conservation by generating an alignment of all currently available vertebrate AMPAR paralogs (>100 sequences). Overall, the NTD is the most divergent segment of the receptor, with a sequence identity score of 52–61% between the paralogs. This is in stark contrast to the conserved LBD/ion channel portion, which shows >80% sequence identity. Within the NTD, the upper lobe (UL) interface is most highly conserved, indicating that contacts mediated by this segment are of functional importance. However, two UL interface positions featured a subunit-specific pattern of conservation: N54 at the upper edge and T78, which forms a polar contact in the core of the interface (Figure 2A and B). These residues are conserved between GluA2–4, whereas in GluA1, position 54 is occupied by a moderately conserved tyrosine and position 78 by a conserved methionine (Figure 2A and D). The fact that these two positions show systematic alteration between assembly partners and localize to the assembly interface indicates that they may be important for the specificity of interaction (Lichtarge et al, 1996). Therefore, we reasoned that these two ‘hotspots’ might encode critical assembly determinants. 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. Download figure Download PowerPoint We first tested the impact of NTD dimer contacts on the stability of the whole receptor. Recent structural insights reveal that NTD associations are prominent contact points within the receptor and NTD dimers are major assembly interfaces in AMPAR dimers (Sobolevsky et al, 2009; Nakagawa, 2010; Shanks et al, 2010). Guided by the structure and our evolutionary analysis, we designed mutations in the assembly-critical GluA2 NTD interface. Blue native (BN) PAGE resolved full-length GluA2, expressed in HEK 293T cells, into stable mono-, di-, and tetrameric species (Figure 3A; Greger et al, 2003); this pattern was altered drastically by mutation. For example, F82A substantially weakened the tetrameric complex (Figure 3A, lane 8; see also Figure 2B). This is to be expected, as targeting the conserved hydrophobic core of the interface will destabilize the receptor non-specifically (Lichtarge et al, 1996). Similarly, targeting the variable hotspots markedly altered tetramer stability (Figure 3A, lanes 2–5, 7). Mild SDS treatment (1% SDS for 10 min at 30°C), which dissociates tetramers and thus facilitates a more direct assessment of dimer stability (Penn et al, 2008), uncovered bidirectional differences for the two hotspots, whereas for N54A, monomers were below detection limit and T78A displayed a five- to sixfold greater fraction of monomers relative to wild type (WT; Figure 3B). These opposing dimer stabilities suggest that mutation at N54 (stabilizing) and T78 (destabilizing) may affect assembly by mechanistically different routes, which in turn may underlie assembly specificity. 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). Download figure Download PowerPoint Functional scanning of the GluA2 NTD dimer interface To extend our findings to heteromeric assembly, we employed a sensitive functional assay to characterize the determinants in the NTD dimer interface. We focused on GluA1/2, which are prominent heteromers throughout the brain and are the major AMPAR species in hippocampus (Lu et al, 2009). Guided by the structure, we introduced mutations into GluA2, which were co-expressed with GluA1 in HEK293T cells. The extent of GluA1/2 heteromerization was assayed by measuring current/voltage (I/V) relationships for an expression level of GluA2 giving optimal dynamic range (Supplementary Figure S3A). GluA1 homomers display inward rectification at positive holding potentials, reflecting blockade of the channel pore by intracellular polyamines. This block is alleviated by co-assembly with GluA2 edited at the Q/R site (Supplementary Figure S3B; Isaac et al, 2007). Thus, an increase of the rectification index (RI), computed as the ratio of slope conductance at +10 and −40 mV (g+10/g−40), indicates increased heteromer formation. We first targeted the lower lobe (LL) of the NTD—residues in the LL interface are identical between GluA1 and GluA2. The inner face of helix E, including L137 and L144, forms a hydrophobic patch (Figure 2C). Breaking these hydrophobic contacts via the L137A/L144A double mutant elevated the heteromerization competence by 2.6-fold (Table II). In addition Q141A, which will abrogate a polar link with N158 and Y131, facilitated heteromerization (3.1-fold; Table II). Weakening the GluA2 LL interface, therefore, increased co-assembly with GluA1. Table 2. Heteromerization of GluA1/GluA2 NTD mutants GluA1 GluA2 Rectification index (RI) Fold change in heteromer assembly competenceb Reversal potential (Erev) n Average LBa UBa Mean s.e.m. WT — 0.027 0.025 0.029 — — — 4 WT WT 0.097 0.085 0.111 1.0 6.1 0.5 20 WT F50A F82A 0.079 0.060 0.104 0.8 5.7 0.8 6 WT F50A F82A L310A 0.041 0.026 0.065 0.3 2.7 0.9 6 WT F82A L310A 0.035 0.023 0.053 0.2 4.5 0.7 10 WT L310A 0.173 0.114 0.262 2.2 4.9 1.0 5 WT T53A 0.113 0.092 0.138 1.2 3.5 1.2 7 WT N54A 0.172 0.127 0.233 2.2 5.4 0.4 8 WT T78A 0.280 0.223 0.352 4.7 2.8 1.4 6 WT S81A 0.138 0.107 0.177 1.6 5.6 0.5 7 WT T53A T78A 0.137 0.114 0.166 1.6 3.7 0.9 10 WT N54A T78A 0.127 0.103 0.157 1.4 5.0 1.2 6 WT N54Y T78M 0.056 0.041 0.078 0.5 5.6 1.6 6 M78T WT 0.230 0.178 0.299 3.4 5.9 0.8 13 Y54N WT 0.207 0.166 0.258 2.9 5.0 0.1 9 M78T Y54N WT 0.171 0.128 0.228 2.2 3.2 1.0 4 WT T78I 0.041 0.026 0.063 0.3 5.4 0.7 5 WT T78L 0.110 0.076 0.159 1.2 5.2 0.4 3 WT T78M 0.109 0.089 0.134 1.2 5.3 0.1 7 WT T78V 0.183 0.141 0.239 2.4 6.3 0.5 5 M78T T78A 0.169 0.120 0.238 2.1 5.4 0.6 6 M78T T78M 0.171 0.126 0.231 2.2 4.6 0.8 6 M78T T78V 0.143 0.099 0.207 1.7 7.1 1.3 8 M78T T78A T53A 0.120 0.086 0.166 1.3 5.8 0.9 9 WT L137A L144A 0.191 0.145 0.253 2.6 5.4 0.6 6 WT Q141A 0.216 0.147 0.318 3.1 6.2 1.2 5 Abbreviations: LB, lower bound; UB, upper bound. a See Materials and methods. b Heteromer assembly competence is defined as the equivalent wild-type GluA2/1 expression ratio required to give mutant rectification index. Determined from the titration curve in Figure S4C. Surprisingly, mutation of most interfacing side chains in the UL, also further increased heteromerization with GluA1, seen by the larger outward currents at positive potentials (Figure 4D; Table II) and summarized by conductance–voltage (G–V) relationships (Figure 4B). It appears therefore that the NTD has not evolved to selectively facilitate heteromeric assembly, but rather to balance heteromerization together with homomerization. We observe a reduction in GluA1/2 heteromer formation only when targeting the hydrophobic core (F50, F82, L310) of the UL interface (Figure 4C and E), which is highlighted for the F82A/L310A (FL) double mutant (Figure 4A–C). These mutants were also destabilized on BN-PAGE (Figure 3A; and data not shown), likely reflecting the non-specific disruption of NTD-mediated associations, both for GluA2/2 homomers and GluA1/2 heteromers. Mutation of most other residues scattered throughout the UL interface facilitated heteromerization (Figure 4E; Table II). 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 geo" @default.
• W2113670233 created "2016-06-24" @default.
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• W2113670233 date "2011-02-11" @default.
• W2113670233 modified "2023-10-16" @default.
• W2113670233 title "Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers" @default.
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• W2113670233 doi "https://doi.org/10.1038/emboj.2011.16" @default.
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