Auxiliary subunits keep AMPA receptors compact during activation and desensitization
AMPA-type glutamate receptors are found at excitatory synapses throughout the mammalian brain , where they convert glutamate release into membrane depolarization . Their fast kinetics ( Colquhoun et al . , 1992; Geiger et al . , 1995; Taschenberger and von Gersdorff , 2000 ) , as well as the physical attributes of synapses ( Xu-Friedman and Regehr , 2003 ) , allow them to follow glutamate transients at rates above 100 Hz . However , the structural dynamics underlying their rapid signaling are unclear . AMPA receptors are tetrameric ligand-gated ion channels . Ignoring their intracellular regions , they consist of three layers formed from distinct domains ( Figure 1A ) . The two extracellular layers assemble from amino-terminal domains and ligand-binding domains , ATDs and LBDs , respectively . These adopt local dimer pairs in resting and active receptors . These dimers pack around a central twofold symmetry axis that switches into fourfold symmetry in the transmembrane ion channel layer . Although the ATD dimers associate with high affinity ( KD nM to μM ( Zhao et al . , 2017 ) , the association of ATD dimers into a tetramer as well as of LBDs into dimers and tetramers is too weak to measure . In case of the LBDs , this weak association has direct functional consequences , because the LBD intra-dimer interface ruptures upon desensitization ( Sun et al . , 2002; Armstrong et al . , 2006; Dürr et al . , 2014; Twomey et al . , 2017b ) . The crowded and narrow synaptic cleft is scarcely wider than the receptors are tall themselves and has narrow edges ( Zuber et al . , 2005; Tao et al . , 2018 ) . This observation suggests that conformational dynamics of the receptor domains and their relation to synapse dimensions has implications in both health and disease . For example , if the extracellular layer of AMPA receptors can undergo large conformational changes rapidly , that is on the millisecond timescale of fast excitatory transmission , this could disrupt possible interactions of the extracellular domains with the pre- and postsynaptic anchoring proteins ( Elegheert et al . , 2016 ) . Activity-dependent anchoring might be a way to regulate synaptic strength ( Constals et al . , 2015 ) . On the other hand , slow rearrangements could be relevant for trafficking , and in disease states . Advances in the structural biology of ionotropic glutamate receptors ( iGluRs ) have produced a catalogue of static conformational snapshots . Several agonist-bound structures ( Nakagawa et al . , 2005; Dürr et al . , 2014; Meyerson et al . , 2014 ) suggest that local dimers in the ATD and LBD layer move apart from each other substantially . This movement away from the central twofold symmetry axis results in an ‘open’ extracellular layer , with the tops of the ATD and LBD dimers splaying wide open ( desensitized structure in Figure 1A ) . The timescale of this broad lateral movement is unknown , because the structural experiments necessarily took place over hours . We therefore set out to investigate the conformational range of agonist-bound AMPA receptors with the aim of distinguishing frequently-visited , short-lived conformations from the long-lived ones that likely have less direct relevance to synaptic transmission . Previously , we demonstrated disulphide bonds and metal bridges trapping receptors in compact , as opposed to ‘open’ LBD arrangements ( Salazar et al . , 2017; Baranovic et al . , 2016; Lau et al . , 2013 ) . To measure the separation of domains in this work , we used bifunctional methanethiosulfonate cross-linkers ( bis-MTS ) of defined lengths ( Loo and Clarke , 2001; Guan et al . , 2002; Armstrong et al . , 2006; Tajima et al . , 2016 ) ( Figure 1D ) . These cross-linkers show specific combination with two free thiol groups , provided by cysteine residues that we engineered . The selective reactivity of a series of probes can in principle report distances , giving them the property of nanometre-scale molecular rulers . Based on activated and desensitized state structures , we reasoned that , crosslinking between ligand binding domain dimers should be state-dependent . Short crosslinkers should be favored in active states with compact LBD layers , and longer crosslinkers should be favored in desensitized states . Our results suggest that the more opened-up a given conformation of the extracellular layer of the receptor is , the slower it is to access . Once attained , these conformations are stable . However , auxiliary subunits restrict the conformational ensemble , maintaining more compact arrangements of the LBD layer , without much separation of the two LBD dimers . This kinetic classification suggests AMPA receptors at synapses probably have similar , compact geometries regardless of their instantaneous gating state .
The rupture of the LBD intra-dimer interface is a structural hallmark of AMPA receptor desensitization , as shown by biophysical studies based on the structures of isolated ligand binding domains ( Sun et al . , 2002; Armstrong et al . , 2006 ) . Several cryo-electron microscopy ( cryo-EM ) structures of full-length receptors ( Nakagawa et al . , 2005; Dürr et al . , 2014; Meyerson et al . , 2014 ) , such as the one shown in Figure 1A , suggest that desensitization might involve further rearrangements of the ligand-binding domains , including movement of the two LBD dimers away from the central twofold axis of symmetry and an even more substantial ‘opening’ of the ATD-layer parallel to the membrane plane . We attempted to capture this movement between LBDs with bis-MTS cross-linkers ranging from 7 to 18 Å in length ( Figure 1D ) . If the LBD layer is opened up in the horizontal plane upon receptor desensitization , this movement should create access for bis-MTS cross-linkers into the inter-dimer space ( orange spheres in Figure 1A ) . Because bis-MTS cross-linkers are flexible ( apart from bMTSp , which is rigid ) , they can bend and bind to Cys residues whose separation is shorter than the length of the cross-linker . Here , we assume that a fully reacted cross-linker will exert its effect mainly by preventing the engineered residues from separating more than the extended length of the cross-linker ( Figure 1D ) . Conversely , if the two free Cys residues never separate beyond the length of the cross-linker during gating , then the cross-linker should have little or no effect . The ability of the cross-linkers of different lengths to access the inter-dimer space in the LBD layer reports on the lateral separation of the two LBD dimers , but does not inform the movements the LBD layer undertook to arrive at the specific conformation . We can assume that the membrane-bound TM region somewhat restricts vertical displacement of the LBDs . Thus , the movements we are probing are those approximately parallel to the membrane . The cross-linkers could in principle be applied to the ATDs , but ATD layer is functionally silent ( Pasternack et al . , 2002 ) and its cross-linking does not produce measurable changes in the receptor activity ( Yelshanskaya et al . , 2016 ) . We , therefore , do not make any extrapolations here about the ATD movements from cross-linking the LBD layer , but structural models and fluorescence studies ( Shaikh et al . , 2016 ) so far indicate that the ATDs do follow movements of the LBD layer ( Figure 1A–B ) . Based on the structural models of homomeric , full-length GluA2 receptors , we identified positions 665 and 666 ( in the loop connecting α-helices F and G in the LBDs ) as best positioned to follow separation of the LBD dimers ( Figure 1A–C ) . The presumptive geometries of the sulfhydryl groups ( SG ) for cysteine mutants at these sites , in the agonist bound states of GluA2 receptor ( with and without auxiliary subunits ) are shown in Figure 1C . The structure of the desensitized GluA2 receptor ( EMDB: 2688 , Figure 1A ) is not detailed enough to measure residue distances , but the equivalent residues are 21 Å apart in homologous kainate receptors ( PDB: 5KUF; Meyerson et al . , 2016 ) . Based on these structural data , all six bis-MTS cross-linkers should be able to modulate desensitized V666C and A665C receptors by preventing engineered Cys at both sites from reaching their full separation in the desensitized state , in the absence of auxiliary subunits . Given the distances in published structures , one might expect M10M to stabilize active V666C receptors in the absence of auxiliary subunit Stargazin and M8M in its presence ( Figure 1C–D ) . As shown in Figure 2B and D , bis-MTS cross-linkers M1M to M10M all caused strong reduction of the peak current in V666C mutant when applied in the desensitized state . To quantify this effect , the peak current was measured from the control pulses before ( Ipeak pre-trap , four pulses ) and after the 1 min application ( trap ) of the cross-linker ( Ipeak post-trap , second control pulse after the trap , arrows in Figure 2A–C ) . For each patch , the ratio of Ipeak post-trap over Ipeak pre-trap was determined and plotted as shown in Figure 2D–E . For the V666C mutant , bis-MTS cross-linkers from 7 to 15 Å in length ( M1M-M8M; 1 µM ) , inhibited ~90% of the peak current in the patch after a 1-min application ( Figure 2—source data 1 ) . The longest ( M10M ) cross-linker was the slowest one to act ( Figure 2F ) , leading to less inhibition ( ~70% ) in the first minute of exposure . The reduction was less pronounced for A665C mutant for all cross-linkers ( ~50% , Figure 2E and Figure 2—source data 1 ) . The slow reaction time of bis-MTS cross-linkers ( ≥minute , Figure 2F ) is a direct consequence of their low concentration ( 1 μM ) . We chose a low concentration in order to minimize the linking of these highly reactive compounds to each other ( Kenyon and Bruice , 1977 ) . However , the slow action of bis-MTS cross-linkers could also be due to the sampling of slowly attained conformations . To determine that modification of the V666C receptors by MTS cross-linkers can proceed on the same time scale as receptor gating , we performed additional trapping experiments with 50 μM M3M and M10M ( Figure 3 ) . If the slow reaction rate of bis-MTS is primarily due to their low concentration , the reaction time should increase accordingly at higher concentrations . Indeed , at 50 μM , both bis-MTS cross-linkers were roughly 50x faster to modify the receptors ( τM3M = 0 . 2 s and τM10M = 1 . 7 s , Figure 3B and D ) . The relative modification time was preserved with the longer cross-linker ( M10M ) still being slower than the shorter one ( M3M ) . These experiments indicate that bifunctional MTS reagents are capturing rapid transitions in receptor structure on the millisecond timescale . Inhibition was overall so profound that we sought to establish that it was specific . Two other factors potentially contribute to the current decrease: non-specific run-down of the current and disulphide bonding of the introduced cysteines to each other . Current run-down is particularly difficult to avoid in the long records that we made for these experiments . A665C and V666C sulfhydryl groups were both previously shown to crosslink in the presence of oxidizing agent CuPhen ( Salazar et al . , 2017; Lau et al . , 2013; Yelshanskaya et al . , 2016 ) . To account for these confounding factors , we made paired recordings: a patch was first exposed to a 1-min long trapping pulse containing glutamate only ( no cross-linkers ) , followed by trapping of the same patch in glutamate and a cross-linker ( Figure 2B–C ) . Any run-down in the patch or possible cross-linking of the cysteines to each other was then assessed from trapping in glutamate only . In case of A665C , the introduced cysteines linked to each other during long exposures to 10 mM glutamate , resulting in the peak current reduction of 0 . 62 ± 0 . 04 ( n = 23 , p < 10−7 vs . WT: 0 . 97 ± 0 . 03 , n = 30 ) , consistent with previous results ( Lau et al . , 2013; Yelshanskaya et al . , 2016 ) . In reducing conditions , the trapped A665C receptors recovered within seconds ( grey dots in Figure 2C , τ = 3 . 3 ± 0 . 4 s , n = 17 ) . V666C mutant underwent less peak current inhibition during exposures to 10 mM glutamate ( V666C: 0 . 86 ± 0 . 02 , n = 44 , p = 0 . 003 vs . WT ) and showed no evidence of recovery in reducing conditions . For V666C mutant , all six bis-MTS cross-linkers resulted in more current inhibition than glutamate alone ( p ≤ 0 . 03 with and without bis-MTS , depending on the cross-linker; paired randomization test ) . Overall , paired recordings gave indistinguishable results to the non-paired recordings . We therefore pooled the cross-linking data in glutamate alone across conditions for each mutant ( Figure 2D–E and Figure 2—source data 1 ) . To confirm that the observed peak current reduction came from crosslinking rather than monofunctional engagement of a cross-linker , we modified mutants with MTSEA ( Figure 1D ) , which can interact only with a single cysteine . As shown in Figure 2D–E , MTSEA failed to inhibit either V666C or A665C above Glu-only control ( V666C: 0 . 74 ± 0 . 008 , n = 4 , p = 0 . 1; A665C: 0 . 81 ± 0 . 06 , n = 4 , p = 0 . 06 ) . Thus , bifunctional MTS cross-linking was necessary for the peak current reduction observed in desensitizing AMPA receptors . The effect of all bis-MTS cross-linkers with respect to their length is summarized in the trapping profile for desensitized V666C receptors ( Figure 2G ) . The profile is a shallow parabola with strong effects across all tested lengths . Fitting a parabola to these data is justified by the observation that direct disulphide crosslinking ( representing the short distance limit ) is quite ineffective at this site ( Figure 2D and Lau et al . , 2013 ) . These results demonstrate that limiting V666C separation of desensitized AMPA receptors to 18 Å causes inhibition , indicating further separation of the residues is possible , in good agreement with cryo-EM structures ( Meyerson et al . , 2014; Dürr et al . , 2014 ) . In addition to positions 665 and 666 , we also tested nearby positions 662 and 664 for their sensitivity to bis-MTS cross-linkers ( Figure 2—figure supplement 1 ) . The I664C mutant showed similar levels of the peak current reduction to V666C . However , structural models position V666C sulfhydryls at distances that are better matched by the length of bis-MTS cross-linkers ( Figure 1C–D ) than the separation of I664C sulfhydryls across different functional states ( Figure 2—figure supplement 1A ) . Like the A665C mutant , the S662C mutant showed considerable disulphide formation . For these reasons , we focused on the V666C mutant in further investigations . Next , we tested if the cross-linkers specifically target introduced cysteines . We utilized the established phenomenon that restraining the LBD intra-dimer interface leads to block of desensitization ( Sun et al . , 2002; Armstrong et al . , 2006 ) . If the bis-MTS cross-linkers specifically target intra-dimer interface , then introducing a single cysteine mutant , positioned within LBD dimers ( Figure 2—figure supplement 2A ) , such as K493C ( Armstrong et al . , 2006 ) , should lead to a block of desensitization depending on the length of the cross-linker . Sulfhydryl groups of K493C residues in resting and active GluA2 receptors with intra-dimer LBD interface intact are 7 and 6 Å apart , respectively ( as measured in the following resting: 3KG2 , 4U2P , 5L1B and active structures: 4UQ6 , 5WEO , using the most common rotamers ) . The desensitized structure in complex with the auxiliary subunit GSG1L has K493C residues 12 Å apart ( 5VHZ ) . It has been previously shown that sulfhydryl groups of residues K493C , in the absence of auxiliary subunits , must separate more than 13 Å for the LBD dimers to desensitize ( Armstrong et al . , 2006 ) . Based on these numbers , cross-linkers that are ≤ 13 Å in length ( M1M , M3M , bMTSp and M6M ) are expected to block desensitization . When applied on K493C receptors ( 1 μM , for ≥ min ) , bis-MTS cross-linkers had profoundly different effect from the mutants at the inter-dimer interface: they potentiated rather than inhibited the current ( Figure 2—figure supplement 2B–D ) . M1M blocked desensitization , as expected , although not completely ( Isteady-state/Ipeak = 49 . 6 ± 4% , n = 4 , p = 0 . 006 vs . K493C desensitization levels in the absence of bis-MTS: 26 . 6 ± 2% , n = 50 ) . At 9 Å , M3M was the most effective in blocking desensitization ( Isteady-state/Ipeak = 80 . 8 ± 2% , n = 16 , p < 10−7 vs . bis-MTS-free desensitization ) . M6M cross-linker blocked desensitization , as predicted ( Isteady-state/Ipeak = 63 . 7 ± 7% , n = 9 , p < 10−7 vs . bis-MTS-free desensitization ) , but rigid bMTSp at 12 Å failed to block desensitization ( Isteady-state/Ipeak = 22 . 3 ± 1% , n = 7 , p = 0 . 5 vs . bis-MTS-free desensitization ) . As expected , none of the longer cross-linkers ( M8M and M10M ) reduced desensitization compared to the non-treated K493C receptors ( M8M: 18 . 3 ± 3% , n = 9 and M10M: 40 . 3 ± 6% , n = 3 , p 0 . 1 for both ) . Although bMTSp and M8M did not reduce desensitization , they both potentiated K493C currents ( they increased steady-state and peak current to the same extent , Figure 2—figure supplement 2B ) , unlike M10M cross-linker , which produced no change in K493C currents . The absence of the effect for M10M could be either because it is too long to bind to K493C receptors or because , even when bound , its length does not restrict movements of desensitizing LBD monomers at position 493 . The bis-MTS length-dependent block of desensitization in K493C receptors strongly indicates the cross-linkers target introduced Cys mutations selectively . We also considered the possibility that bis-MTS cross-linkers might be spuriously cross-linking to wild-type cysteines on the receptor or forming inter-receptor cross-links ( Figure 2—figure supplement 3A ) . If this were the case , the peak current reduction effect would be expected to scale with the number of the receptors in the membrane , that is peak current , but no such correlation was found ( Figure 2—figure supplement 3B ) . In addition , longer cross-linkers would be expected to be more efficient in forging inter-receptor cross-links , but we found that the longest cross-linker , M10M , was the slowest to react on desensitized AMPA receptors ( Figure 2F ) . The absence of the strong peak current reduction in the WT receptors also speaks against the bis-MTS cross-linkers interacting with native cysteine residues . These results led us to conclude that the bis-MTS cross-linkers can cross-link cysteines introduced at the LBD inter-dimer interface ( V666C ) . Upon establishing specific and strong reduction of the peak current in desensitizing V666C receptors by the bis-MTS cross-linkers , we next sought to examine the stability of trapped states . The more stable the trapped state , the longer we would expect that it takes for trapping effects to reverse , and vice versa . The fastest recovery after trapping was observed with M1M ( τ = 30 ± 7 s , n = 5 ) and the time constant of the peak current recovery could be measured by directly fitting the peak current of the post-trap control pulses ( grey dots in Figure 2B ) . With longer bis-MTS cross-linkers , the recovery time increased to minutes , making direct measurements of the recovery time from post-trap control pulses impractical . Instead , the experimental design was adjusted to allow measurements of long recovery times as described in Materials and methods and Figure 4 . After the receptors were trapped with M3M for 1 min , the peak current in the patch did recover , but very slowly , taking over 10 min ( 600 applications of Glu ) to reach the pre-trapping levels . With longer cross-linkers , the peak current was essentially irreversible over the timescales we could measure ( 20 min; Figure 4D–G ) . The progressive inability of the receptors to recover from trapping with longer cross-linkers indicates increasingly stable trapped states corresponding to greater separation of the cross-linked subunits in the desensitized state ( Figure 4G–H ) . Desensitized AMPA receptors with a disulphide bridge formed between the two V666C residues at inter-dimer interface fit in well with this trend , recovering in seconds following 100 s exposure to the oxidizing agent CuPhen ( Figure 4H ) . Multiple stable desensitized states trapped by bis-MTS are a possible explanation for the low-resolution cryo-EM structures of desensitized AMPA receptors in the absence of auxiliary subunits ( Meyerson et al . , 2014; Dürr et al . , 2014 ) . To ensure that the lack of recovery was not due to a limited reducing capacity , we tested a higher concentration of the reducing agent DTT ( 5 mM instead of 1 mM ) . Stronger reducing conditions did not consistently promote recovery of V666C receptors trapped by a long cross-linker M6M ( 12 Å; p = 0 . 3 , Figure 4—figure supplement 1 ) . We next investigated if the extracellular layer of activated V666C receptors is also accessible to a similarly wide range of bis-MTS cross-linkers . According to the cryo-EM structure of AMPA receptor in complex with glutamate and a desensitization blocker ( Meyerson et al . , 2014 ) ( Figure 1C ) , V666C residues are 19 Å apart and therefore we reasoned that all six cross-linkers should restrain the receptor with M10M ( 18 Å ) expected to stabilize the active conformation . To maintain the active state , we blocked desensitization with cyclothiazide ( CTZ , 100 μM ) . As shown in Figure 5 , block of desensitization reduced inhibition by bis-MTS cross-linkers in the first minute of exposure ( see Figure 5 – source data 1for statistics ) . Current inhibition for every cross-linker in the presence and absence of desensitization is shown in Figure 5B . With M1M , about 15% of V666C receptors recovered from inhibition with a time constant of τ = 7 . 1 ± 1 s , n = 4 , leading to the final inhibition of 0 . 65 ± 0 . 02 , n = 12 ( not shown ) . For M3M , the recovery was still faster with τ = 1 . 98 ± 0 . 2 s , n = 8 ( grey dots in Figure 5A ) , with about 15% the receptors recovering and leading to the final inhibition of 0 . 53 ± 0 . 03 , n = 9 . The fast recovery indicates that with dimer separation of about 9 Å , active receptors were trapped in an unstable , stereochemically strained state . Notably , the SG of V666C are 9 Å apart in a structure solved with the partial agonist NOW , which may represent a pre-open state ( Yelshanskaya et al . , 2014 ) . With desensitization blocked , AMPA receptors displayed a distinct trapping profile from that of desensitized receptors ( Figure 5C ) . All cross-linkers modulated active state less than the desensitized in the first minute of exposure , apart from M8M ( 15 Å; Figure 5B ) and a fraction of active receptors trapped with the shorter cross-linkers ( M1M and M3M ) recovered fast from the trap . The trapping profile against linker length was sharper , indicative of less conformational heterogeneity . At 12–15 Å ( bMTSp , M6M and M8M ) , active receptors were trapped most effectively and without fast recovery; at 18 Å ( M10M ) the extent of trapping decreased ( Figure 5—source data 1 ) . This places the preferred V666C sulfhydryl separation in the active state around 15 Å , somewhat closer than 19 Å obtained in the cryo-EM structure of active GluA2 ( Figure 1C ) . The description of the trapping profile by a parabola ( with minimum at 13 Å ) was not as good as for the desensitized condition , with the profound trapping by M8M being underestimated . The generally reduced inhibition of active receptors , compared to desensitized ones , led us to investigate the possibility that bis-MTS reagents modify non-desensitizing V666C receptors in other ways than current amplitude reduction . We measured the rate of receptor deactivation before and after the trap in M10M in the presence of desensitization blocker CTZ and found no difference ( Figure 5—figure supplement 1C–D , τpre-trap = 1 . 7 ms ± 0 . 2 , τpost-trap = 1 . 6 ± 0 . 2 ms , n = 8 , p = 0 . 05 ( paired randomization test ) ) . We considered the possibility that non-desensitizing ( CTZ bound ) receptors were modified by M10M in a functionally silent manner . We tested this scenario with the following experiment: a patch with V666C receptors was first trapped in M10M and CTZ; CTZ was then washed-out of the patch until V666C receptors were able to desensitize again freely ( Figure 5—figure supplement 1A ( 2 ) ) . After this , we exposed receptors to M10M and glutamate ( Figure 5—figure supplement 1A ( 3 ) ) . If non-desensitizing V666C receptors had been silently modified by M10M , then a fraction of the receptors should have been protected resulting in the reduced sensitivity to further trapping by M10M . V666C receptors initially exposed to M10M in the presence of CTZ were modified to the same extent as naïve receptors ( Ipeak post-trap/Ipeak pre-trap for V666C initially trapped in CTZ: 0 . 23 ± 0 . 04 , n = 6 and for V666C never trapped in CTZ: 0 . 30 ± 0 . 03 , n = 16 , p = 0 . 07; Figure 5—figure supplement 1B ) . Taken together , these results strongly suggest that non-desensitizing V666C receptors were not silently modified by M10M . Instead , the reduced inhibition of active receptors reflected state-dependent protection from modification . The effect of M10M was inhibitory rather than potentiating , despite its length ( 18 Å ) matching closely the predicted V666C separation of 19 Å ( Figure 1C ) . Trapping with bis-MTS cross-linkers so far indicated more conformational flexibility of the LBD layer in desensitized than activated AMPA receptors . However , synaptic AMPA receptors are rarely expressed alone and are instead associated with various auxiliary proteins that re-define their kinetic properties ( Schwenk et al . , 2012; Jackson and Nicoll , 2011 ) . We therefore wondered if the presence of auxiliary subunits , such as Stargazin ( Stg ) , could affect conformational flexibility of AMPA receptors . Recently , cryo-EM structures of AMPA receptors in complex with auxiliary subunits have been acquired in resting , active and desensitized states ( Figure 1B ) ( Chen et al . , 2017; Twomey et al . , 2017a ) . These structures all estimate reduced separation of V666C residues when compared to receptors without auxiliary proteins . For example , sulfhydryl groups of V666C residues on subunits A and C are 19 Å apart in the activated receptors without Stg and 15 Å in complex with Stg ( Figure 1C ) . In the desensitized state , the equivalent residues are 21 Å apart in homologous kainate receptors ( PDB: 5KUF [Meyerson et al . , 2016] ) and 17 Å in desensitized GluA2 receptors associated with GSG1L auxiliary proteins ( Figure 1C , PDB: 5VHZ [Twomey et al . , 2017b] ) . If auxiliary subunits keep the LBD layer more compact , we reasoned that their presence should also limit the effects of longer bis-MTS cross-linkers . For example , the longest cross-linker ( M10M ) could be expected to have less effect on the desensitized V666C receptors in the presence of Stg . To test this hypothesis , we repeated the trapping experiments on complexes of AMPA receptors with Stg ( Figure 6 ) . GluA2 and Stg were co-expressed and association of complexes was assessed by measuring the ratio of kainate current over glutamate current ( KA/Glu ) . The relative efficacy of the partial agonist kainate is known to be higher for GluA2-Stg complexes than for GluA2 alone , making it a good marker of GluA2-Stg association ( Tomita et al . , 2005; Shi et al . , 2009 ) . After establishing formation of the GluA2 V666C-Stg complexes in the patch , we proceeded with the trapping protocol that exposed complexes to glutamate and a bis-MTS cross-linker ( 1 μM ) for 1 min ( Figure 6A–B ) , as described previously . No blocker of desensitization was added and the receptors were allowed to desensitize freely . Stargazin reduces desensitization of AMPA receptors so the crosslinking represents trapping across a mixture of active and desensitized states . The trapping results summarized in Figure 6C show that the presence of auxiliary subunit Stg apparently protected V666C receptors from cross-linking by bis-MTS . Indeed , following trapping , a robust response was preserved , and could not be overcome by longer trapping intervals ( Figure 6—figure supplement 1A–B ) . Based on published structures , the decrease in the trapping extent was expected for M10M ( Figure 6C and Figure 6—source data 1 ) , but not for shorter bis-MTS . This result indicates the presence of alternative conformations that have a more compact extracellular layer or that otherwise render cysteines inaccessible to modulation by bis-MTS . To test whether bis-MTS cross-linkers perhaps act on non-complexed V666C receptors only , without affecting V666C-Stg complexes , we measured the KA/Glu current ratio before and after the bis-MTS trap for a series of patches . We reasoned that if only non-complexed V666C receptors were being modified , the glutamate-activated current should reduce , but the kainate current ( which is almost entirely carried by GluA2-Stg complexes ) should not . Therefore , preferential trapping of non-complexed V666C mutants should lead to an increase in KA/Glu ratio . As shown in Figure 6E , the KA/Glu ratio was not affected ( before trap: 0 . 46 ± 0 . 03; after trap: 0 . 43 ± 0 . 02 , n = 13; p = 0 . 2 , paired randomization test ) , indicating V666C-Stg complexes were being modified by bis-MTS cross-linkers . The trapping profile of V666C-Stg complexes ( orange in Figure 6D ) reflects the partial protection from trapping in the presence of Stg for all cross-linker lengths , but its overall shape is practically superposable onto the trapping profile of desensitized V666C receptors without Stg ( red , dashed line in Figure 6D ) . Strikingly , the two curves reach their minimum at the same point of 11 Å ( triangles in Figure 6D ) and have identical curvature . This indistinguishable length dependence indicated that the trapping of V666C-Stg complexes came primarily from trapping desensitized receptors , and that the active complexes of V666C-Stg might be untouched by bis-MTS cross-linkers . To test this hypothesis , we repeated the trapping protocol in the presence of CTZ , to block desensitization of V666C-Stg complexes . Two cross-linkers were tested in this condition: M1M , the shortest one , and M8M , which had one of the strongest trapping effects on active V666C receptors without Stg ( Figure 5B–C ) . As predicted , neither bis-MTS cross-linker had any effect on activated GluA2-Stg complexes ( M1M: p = 0 . 1 , n = 9; M8M: p = 0 . 2 , n = 9; paired recordings with and without bis-MTS ) ( Figure 7 ) . Over very long exposures , M8M could induce irreversible inhibition , presumably from residual desensitization ( Figure 6—figure supplement 1 ) . This result confirmed that partial trapping observed for desensitizing V666C-Stg mainly derived from bis-MTS cross-linkers accessing desensitized complexes . We next wondered if the protection from trapping observed for active GluA2-Stg complexes could , at least partly , be explained by reduced accessibility of cysteines during exposure to bis-MTS , perhaps because the Cys666 residues are buried against other subunits or oriented such that the sulfhydryl groups are inaccessible for cross-linking . We mined possible conformations of the active LBD tetramer ( PDB: 5WEO ) using a coarse docking approach , allowing each active dimer to move in the membrane plane , and allowing rotation of one dimer with respect to the other around axes parallel and perpendicular to the active dimer interface ( Figure 7D and E ) . Interestingly , docked structures with low Cys666 accessibility segregated into two classes , a loose arrangement where Cys666 were physically close but sterically hindering each other ( with the centre of mass separation of the two active dimers comparable to 5WEO , for example models m17 and m32 in Figure 7D ) , and a tighter arrangement of the LBDs with V666C close to helix K ( with the centre of mass separation of the two active dimers smaller than in 5WEO , for example m223 and m265 in Figure 7D ) , not unlike the structure of the V666C mutant LBD bound with fluorowillardine ( 5JEI ) ( Salazar et al . , 2017 ) . In that structure , additional shielding of the V666C side chain is afforded by its being shielded by neighboring structural elements . Thus , docked structures lead us to hypothesize that active GluA2-Stg complexes could avoid cross-linking not just by adopting more compact LBD arrangements , but also by preferring conformations which shield V666C SG groups .
Technical advances in cryo-electron microscopy have revolutionized the study of membrane proteins , and the supply of structural information is greater than ever before . However , as the catalogue of images swells , the need to relate their geometry to dynamics becomes more pressing . In case of AMPA receptors , full-length structures in complexes with various ligands and auxiliary subunits have been published . Here , we have used a classical crosslinking approach allied to rapid perfusion to measure distances up to 18 Å in AMPA receptor extracellular domains across different functional states and in the time domain . Bis-MTS cross-linkers have been used previously to aid identification of movements underlying state transitions in AMPA and NMDA receptors ( Armstrong et al . , 2006; Tajima et al . , 2016 ) . Here , we report effects with a strong dependence on the target site , length of the cross-linker , functional state of the receptor and the presence or absence of auxiliary subunits . Since most bis-MTS cross-linkers are alkyl chains and hence flexible , they can cross-link pairs of Cys residues closer to each other than the extended length of the cross-linker . For example , M10M could cause the same effect when bound to two Cys that are 7 or 15 Å apart , acting as a universal inhibitor for a range of Cys distances . For this reason , we used bis-MTS ranging from 7 to 18 Å in length ( M1M – M10M ) . Our results show an optimum ( inhibitory ) effect of the cross-linkers at position V666C , with M10M not producing as strong effect as shorter cross-linkers . This is inconsistent with the interpretation of the universal inhibitor and suggests bis-MTS cross-linkers exert their effect mainly by preventing the engineered residues from separating more than the extended length of the cross-linker . We also tested a rigid cross-linker , bMTSp , whose length matches closely that of flexible M6M ( 12 and 13 Å , respectively ) . This places a lower limit on separation of V666C Cys at ~12 Å . The only difference we observed between bMTSp and M6M was with intra-dimer K493C mutant , with M6M blocking desensitization more effectively than bMTSp , perhaps indicating an angle between the K493C side chains that was more easily accommodated by the flexible chain . All bis-MTS cross-linkers , when applied to the mutants at the inter-dimer LBD interface , inhibited the current , albeit to a different degree depending on the functional state of the receptors . Whereas cross-linking of the desensitized states should result in current inhibition , cross-linking of an open channel would be expected to result in more active receptors . However , to date all inter-dimer traps have been inhibitory . Inhibition seems independent of the nature of the constraint , whether disulphide bonds ( Plested and Mayer , 2009; Yelshanskaya et al . , 2016 ) , metal bridges ( Lau et al . , 2013; Baranovic et al . , 2016 ) , flexible or rigid bis-MTS cross-linkers ( this study ) . In addition , a structure of the full-length GluA2 receptor carrying a gain-of-function mutation and in complex with a partial agonist , blocker of desensitization and a peptide constraining the LBD dimers resulted in a closed channel ( Chen et al . , 2014 ) . Cysteine crosslinks within dimers inhibit the gating of kainate receptors , even when blocking desensitization ( Daniels et al . , 2013 ) . Whereas the peptide binding , zinc and disulphide bridges only form under strict geometrical requirements , possibly incompatible with a stably open pore , the same cannot be said for bis-MTS cross-linkers . The cross-linkers are flexible and therefore do not restrict the trapped LBD tetramer to a single geometry . The inhibitory effect of the crosslinkers is not universal – at the active intra-dimer interfaces we could potentiate receptors as shown previously ( Armstrong et al . , 2006 ) . Structural models of full-length AMPA receptors in various states of activity do not explain why every restraint of the LBD dimers leads to a decrease in activity . Either the correct sites for potentiation have not yet been found , or this observation reflects a mechanistic detail of receptor activation . The mechanism behind this inhibition remains frustratingly unclear , limiting the interpretation of the cross-linking approach used in this study . In Figure 8A–B , the trapping profiles of V666C receptors with the available structural models of GluA2 in the equivalent condition are compared . The shallow trapping profile of desensitized AMPA receptors indicates a structural ensemble of conformations broadly in agreement with multiple desensitized states ( Meyerson et al . , 2014; Robert and Howe , 2003 ) . Even though the LBD dimer-dimer separation observed in the model ( > 18 Å , EMDB: 2688 ) was not tested with the bis-MTS cross-linker of the corresponding length , extrapolation of the trapping profile indicates such conformations are available to desensitized AMPA receptors . However , the longest cross-linker ( 18 Å ) was the slowest one to act on desensitized receptors , indicating that this conformation , and more ‘open’ ones , are not readily available for cross-linking , but slowly get populated during prolonged exposures to agonist . The longest cross-linker , M10M , was also the slowest one to act in the active state . There , the strongest cross-linking effect was achieved by the 12–15 Å-long bis-MTS ( green in Figure 8B ) . This suggests inter-dimer V666C residues are closer together in the active state than 19 Å , predicted by the structural model ( PDB: 4UQ6 ) . The extracellular layer was also seen in an ‘opened’ arrangement in NMDA receptors ( Zhu et al . , 2016 ) , with the caveat that these structures were antagonist-bound and therefore not equivalent to our observations . The presence of Stg lead to universal attenuation of the cross-linking effect , for all bis-MTS lengths and both functional states , desensitized and active ( orange and blue in Figure 8A and B , respectively ) . The longest crosslinker ( M10M ) produced the least trapping , even following long exposures ( ~3 min ) . Thus , the LBD layer could not be modulated by bis-MTS cross-linkers for majority of these receptors . This result chimes with structural and FRET experiments ( Shaikh et al . , 2016 ) , where the presence of Stg made the LBD and ATD layer more compact . In the desensitized state , the shape of the trapping profile is the same , with or without Stg ( Figure 8A ) , with both parabola reaching a minimum point at 11 Å ( triangles in Figure 8A ) . This is in excellent agreement with the structure of desensitized GluA2-Stg complex ( PDB: 5VOV ) . In the active state with Stg , our results deviate from the available full-length active structures . Both short and long ( 7 or 15 Å ) bis-MTS reagents failed to inhibit currents in the first minute of trapping , indicating more compact LBD arrangements over this timescale than obtained in the structures . In other words , if structures can be obtained in a time-resolved manner in milliseconds following glutamate exposure ( Unwin , 1995 ) , our results suggest they should be more compact than structures solved to date . The protection from trapping in our cross-linking experiments most likely has multiple origins . Although ‘protection’ could result from a persistent long-distance separation , outside the range of the crosslinkers , several observations and common sense speak against this possibility . First , we previously showed that active receptors could be trapped by zinc bridges in compact arrangements ( Baranovic et al . , 2016 ) . Second , the long distance would have to be maintained throughout the exposure , because any transit between compact and dilated arrangements must pass through intermediate separations , allowing crosslinkers to span the gap , assuming extremely rapid reactivity of the bis-MTS compounds ( Kenyon and Bruice , 1977 ) . Our reaction rates are close to the maximum expected ( 105 M–1s–1; Liu et al . , 1997 ) . Third , even the most dilated structures are in the range of crosslinker lengths that we used . Fourth , the mixed trapping condition for GluA2-Stg complexes in the absence of CTZ apparently supports desensitized state trapping over a wide range of geometries but no additional active state trapping . We cannot discount specific , state-dependent protection from crosslinking , produced by a unique , closed-cleft LBD conformation provoked by Stg in the active state , but it seems to us unlikely . We reasoned that protection from crosslinking could , at least partly , be accounted for by reduced accessibility of cysteine residues during exposure to bis-MTS . Docking data in Figure 7D–E show that conformations which reduce the accessibility of the V666C side chains are indeed accessible to the active LBDs . These arrangements are more compact than the available structures of the full-length receptor in the active state ( Chen et al . , 2017; Twomey et al . , 2017a ) . No matter how avid and complete trapping is , a possible source of receptor activity after trapping is the activity of free ( non-crosslinked ) subunits ( Figure 8—figure supplement 1 ) . Based on the GluA2 structural models , bis-MTS compounds cross-link subunits proximal to the central twofold symmetry axis at the LBD level ( subunits A and C ) , while sparing the distal subunits , B and D ( Figure 1A–C ) . In AMPA receptors without Stg , with desensitization blocked , the activity of only two subunits produces subconductance openings with short open times ( Rosenmund et al . , 1998 ) . But the presence of Stg ( Coombs et al . , 2017; Zhang et al . , 2014 ) increases the current carried by receptors with one or two active subunits . In our experiments , Stg had an indefatigable effect of maintaining receptor activity in the limit of long exposures to bis-MTS . The effect of Stg was inordinately large , with a maximum effect of the crosslinking leaving approximately half the receptor activity unscathed . Can the two non-crosslinked subunits ( B and D ) produce this level of activity ? A simple back-of-the-envelope calculation suggests this effect is larger than expected . Single channel recordings of GluA2 with Stg reveal that the conductance from two subunits , with desensitization blocked , should be on average about 40% of a full opening but with Popen < 1 ( Coombs et al . , 2017 ) . Therefore , residual activity of the non-crosslinked subunits is higher than expected , unless the B and D subunits have a predominant role in driving channel gating ( as proposed from structural studies [Sobolevsky et al . , 2009] ) . Physiological activation and desensitization of AMPA receptors takes place on a millisecond timescale . The fast gating contrasts desensitized states trapped by bis-MTS cross-linkers that take tens of minutes to recover . It seems reasonable to assume that the degree of stabilization by different crosslinkers is similar , and that the difference in stability comes from the states themselves . This idea is supported by the cut-off that we observe – for crosslinkers longer than 10 Å , crosslinking in desensitized states is irreversible over 10 min ( also in fivefold higher concentration of the reducing agent ) . It is possible that upon cross-linking , V666C residues adopt conformations that make them inaccessible to the reducing agent , but this would then have to be highly specific for bis-MTS reagents longer than 9 Å and state-specific ( due to much faster recovery rates of active than desensitized receptors after trapping in M1M or M3M ) . In addition , in reducing conditions , AMPA receptors recover from disulphide crosslinking at the same sites in hundreds of milliseconds ( Lau et al . , 2013; Salazar et al . , 2017 ) . Assuming that the length of the bis-MTS cross-linkers reflects level of structural rearrangements and following the trend plotted in Figure 4H , this suggests that AMPA receptors at synapses , exposed to brief glutamate transients and in complex with auxiliary subunits , are unlikely to undergo extreme conformational changes . Considering that all of the experiments presented here were done with over-expressed receptors and auxiliary subunit Stg , it is possible that V666C-Stg complexes had variable stoichiometry of association , depending on the expression level . This could impact how Stg affects conformational dynamics of the receptors , but a detailed investigation of this is beyond the scope of this work . We note , however , that the presence of two copies of auxiliary subunit GSG1L were sufficient to keep the LBD layer compact in structural experiments ( Twomey et al . , 2017b ) . Long agonist exposures of minutes to hours are standard in structural biology experiments and could , thus , contribute to the prevalence of more ‘open’ conformations in full-length structures without auxiliary subunits ( Figure 8C ) . However , long exposures to agonists are at odds with synaptic conditions where AMPA receptors see glutamate on a millisecond timescale , before it is actively cleared by transporters ( Clements , 1996 ) . Furthermore , any large structural rearrangements of the extracellular domains would need to be accommodated by the crowded synaptic environment ( High et al . , 2015; Tao et al . , 2018 ) and potential presynaptic interaction partners ( Saglietti et al . , 2007; Elegheert et al . , 2016 ) . When in complex with auxiliary subunits , no functional state of AMPA receptors necessitates large domain movements , and compact arrangements of the extracellular layer can sustain the gating process . We conclude that extracellular domains of synaptic AMPA receptors are unlikely to undergo large structural rearrangements during synaptic transmission and instead work in a fairly compact conformational regime , unless faced with long exposures to glutamate in pathological conditions .
In all experiments , the unedited ( Q586 ) GluA2flip version of rat GluA2 gene was expressed from the pRK5 vector . Amino acid numbering refers to the mature receptor assuming a signal peptide of 21 amino acids in length . As a marker of transfection , eGFP was expressed from the same vector , downstream from an internal ribosomal entry sequence ( IRES ) . Mouse Stargazin gene ( a kind gift from Susumu Tomita ) was expressed from a separate pRK8 vector containing IRES-dsRed ( Carbone and Plested , 2016 ) . All mutations were introduced by overlap PCR and confirmed by double-stranded sequencing . GluA2 constructs were expressed transiently in HEK293 cells using calcium-phosphate precipitation or PEI method as described previously ( Baranovic et al . , 2016; Riva et al . , 2017 ) . HEK293 cells were obtained from the Leibniz Forschungsinstitut DSMZ ( Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH , Germany ) ACC no . 305 ( RRID: CVCL_0045 ) and tested negative for mycoplasma . Cells were maintained in MEM Eagle medium ( PAN-Biotech GmbH , Aidenbach , Germany ) supplemented with 10% ( v/v ) fetal bovine serum and antibiotics ( penicillin ( 100 U/mL ) and streptomycin ( 0 . 1 mg/mL; PAN-Biotech ) . For transfections , 2–3 μg of DNA was transfected per 35 mm dish and cells were washed after 6–8 hr . Recordings were performed 24–72 hr after the transfection at room temperature . For transfections with Stargazin , Stargazin DNA was co-transfected with GluA2 DNA at 2:1 mass ratio and after the transfection , cell medium was supplemented with 40 μM NBQX to reduce Stargazin-induced cytotoxicity . Chemicals were obtained from Sigma Aldrich ( Munich , Germany ) , Abcam plc ( Cambridge , UK ) and Hello Bio ( Bristol , UK ) . MTS compounds were obtained from Toronto Research Chemicals ( North York , Canada ) . The internal ( pipette ) solution for recordings without Stargazin contained ( mM ) : 115 NaCl , 1 MgCl2 , 0 . 5 CaCl2 , 10 NaF , 5 Na4BAPTA , 10 Na2ATP , 5 HEPES , titrated to pH 7 . 3 with NaOH . For recordings with Stargazin , the internal solution was slightly modified: 120 NaCl , 0 . 5 CaCl2 , 10 NaF , 5 Na4BAPTA , 5 HEPES and 0 . 05 spermine , pH 7 . 3 . The external recording solution in all experiments contained ( mM ) : 150 NaCl , 0 . 1 MgCl2 , 0 . 1 CaCl2 and 5 HEPES , pH 7 . 3 . Different drugs were added to the external solution as needed . Glutamate was always applied at 10 mM and DL-dithiothreitol ( DTT ) at 1 mM . Cyclothiazide ( CTZ ) and kainate ( KA ) were thawed from stock solutions on the day of the experiment . Final CTZ and KA concentrations in all experiments were 100 μM and 1 mM , respectively . All MTS compounds were obtained as powder . Although monofunctional MTS reagents are known to be highly reactive and unstable in aqueous solutions ( Kenyon and Bruice , 1977 ) , this information is lacking for bifunctional cross-linkers used in this study . Hence , we took special care to minimize exposure of bis-MTS compounds to oxidizing ( aqueous ) solutions ( Takatsuka and Nikaido , 2010 ) . The powder was dissolved in DMSO , aliquoted and kept on ice on the day of the experiment . Once a stable patch recording was obtained , an aliquot was dissolved in external solution to a final concentration of 1 μM and applied to the patch . This way , aqueous bis-MTS solutions were on average 2–3 min old at the moment of application . Each MTS stock was tested with GluA2 wild-type receptors and K493C receptors as negative and positive controls , respectively . The final MTS concentration of 1 μM was chosen based on previous work ( Sobolevsky et al . , 2003; Yelshansky et al . , 2004 ) . We avoided higher concentrations of MTS compounds due to potential cross-reactivity and chaining effects . Ligands and drugs were applied to outside-out patches via a custom made 4-barrel glass ( VitroCom , USA ) mounted to a linear piezo–electric wafer ( PiezoMove P-601 . 4 , PI , Germany ) ( Lau et al . , 2013 ) . Two barrels were perfused with control solutions and the third barrel with the trapping solution , as described below . All patches were voltage clamped at −40 mV unless stated otherwise . Currents were low-pass filtered at 10 kHz ( −3 dB cut-off , eight-pole Bessel filter ) using an Axopatch200B amplifier ( Molecular Devices , USA ) and acquired with AxographX software ( Axograph Scientific , Australia , RRID:SCR_014284 ) at 20 kHz sampling rate via Instrutech ITC-18 digitizer ( HEKA , Germany ) . Current traces were digitally filtered at 1 kHz ( low-pass ) for presentation in figures . To assess the effect of different bifunctional cross-linkers on AMPA receptors , the receptors were exposed to the cross-linker ( 1 µM ) for 1 min . Before and after this trapping exposure , the current in the patch was tested with control pulses that contained only 10 mM glutamate , without the cross-linker and in the presence of DTT ( 1 mM ) as a reducing agent ( Figure 2A–C ) . Four control pulses before application of the cross-linker provided a measure of the patch current before any exposure to the cross-linker . Accordingly , ( up to thirty ) control pulses recorded after the MTS application , were used to assess any changes in the patch current imparted by the cross-linker treatment . For recordings of GluA2 receptors co-expressed with auxiliary subunit Stargazin , care must be taken that GluA2 receptors are indeed associating with Stargazin . One strategy to minimize the presence of lone V666C receptors relies on the relief of spermine ( polyamine ) block at positive voltages imparted by complexation with Stargazin ( Carbone and Plested , 2016 ) . Although we have included spermine in the pipette solution and measured relieve of block for each patch , we did not perform recordings at positive voltages , as the currents were not stable enough during minutes-long trapping protocols . Instead , a change in kainate efficacy was used as a marker of GluA2-Stargazin association as described in the text . Trapping effects were quantified as the ratio of the average current after the trap ( determined from the second post-trap control pulse ) and average current before the trap ( determined from the four pre-trap control pulses; arrows in Figure 2A–C ) :Activefraction=IposttrapIpretrap In case of desensitizing receptors , peak current was measured and in the case of non-desensitizing receptors , steady-state current . The trapping time of each cross-linker ( at 1 μM ) was determined from cumulative exposures to a bis-MTS of up to 6 min ( six repetitions of the 1-min trapping protocol ) . After each application , current reduction was determined with respect to the initial current in the patch , before any trap . The resulting current decay was described by a monoexponential fit in Igor Pro ( v7 . 06 , Wavemetrics , Lake Oswego , Oregon , USA , RRID:SCR_000325 ) . This approach most likely overestimates the time M3M and M6M need to trap the V666C receptors , as majority of the current is inhibited within the first minute of exposure to bis-MTS ( Figure 2F ) . For the experiments with 50 μM bis-MTS ( M3M and M10M , Figure 3 ) , where faster trapping rates are expected , the experiment was redesigned to measure the peak current inhibition every 50 ms , rather than in 1-min intervals . To determine the rate of recovery from trapping by MTS cross-linkers , the number of post-trap control pulses was increased until full recovery was attained . An envelope of post-trap peak current responses was then created in Igor Pro and fit with a monoexponential . This approach was possible only for faster recovery rates , on the time scale of seconds , such as recovery of desensitized V666C receptors from trapping with M1M ( Figures 2B and 4D ) and from direct V666C sulfhydryl cross-links ( Figure 4H ) and recovery of active V666C receptors from trapping with M1M and M3M ( Figure 5A ) . With longer bis-MTS cross-linkers , the recovery time increased from seconds to minutes , making direct measurements of the recovery time from post-trap control pulses impractical . Instead , the experimental design was re-adjusted to allow measurements of long recovery times as described in Figure 4A–C . In brief , peak current in the patch was initially recorded with 100 ms jumps into glutamate in control conditions until it stabilized . Then , a trapping protocol was performed as described above , with control pulses before and after the trap . After the trapping protocol , the current in the patch was again monitored , for about 10 min , with fast control jumps into glutamate in order to follow any potential recovery of the peak current . In this time period , desensitized V666C receptors managed to recover only from trapping by M1M and M3M ( Figure 4D–G ) , in which case the recovery was fit with a monoexponential . Trapping profiles ( Figures 2G and 6D ) were fit with a parabola in Igor Pro:fx= K0+K1 ( x-K2 ) 2where K1 defines the curvature , K0 the minimum effect and K2 the x value at the minimum . Data points were weighted by the standard error of the mean for the fit . To investigate structures of the LBD tetramer that could preclude trapping by blocking access to the Cys666 SG moiety , we treated each dimer as a rigid body and subjected them to rotations and translations in the membrane plane . Python scripts ( available at https://github . com/aplested/cystance; copy archived at https://github . com/elifesciences-publications/cystance ) were written as a glue for molecular manipulations in PyMOL and CCP4 ( RRID:SCR_007255 ) functions to measure geometry and exposure of the cysteine ( AREAIMOL , NCONT ( Winn et al . , 2011 ) ) . For each run , trial arrangements that reduced Cys666 SG accessibility in subunit A whilst also keeping the dimers in close proximity ( with minimal atom clashes ) and maintaining physiologically plausible in-plane linker arrangements were retained as seeds for subsequent rounds , and the step size was reduced . Trial arrangements with more than 10 atom clashes ( < 2 . 2 Å ) were rejected . No refinement was done to eliminate spurious clashes from flexible surface residues . The optimization was ended when no further improvement was possible . Each search lasted about 5–10 min on a 2017 Macbook Pro . All p values were determined by non–parametric randomization test ( non–paired , unless stated otherwise ) , using at least 105 iterations ( DC-Stats suite: https://github . com/aplested/DC-Stats ) . Bars in graphs indicate mean and error bars SEM . Structural models and related measurements were visualized and measured in PyMOL ( v2 . 0 , RRID:SCR_000305 ) ( The PyMOL Molecular Graphics System , Version 2 . 0 Schrödinger , LLC ) . The length of bis-MTS cross-linkers was measured in ChemDraw Professional ( PerkinElmer , USA . ) .
Signal transduction at vertebrate excitatory synapses involves the rapid activation of AMPA ( α-amino-3-hydroxy-5-methyl-4-isoxazole propionate ) receptors , glutamate-gated ion channels whose four subunits assemble as a dimer-of-dimers . Technical advances in cryo-electron microscopy brought a slew of full-length structures of AMPA receptors , on their own and in combination with auxiliary subunits . These structures indicate that dimers might undergo substantial lateral motions during gating , opening up the extracellular layer along the central twofold symmetry axis . We used bifunctional methanethiosulfonate cross-linkers to calibrate the conformations found in functional AMPA receptors in the presence and absence of the auxiliary subunit Stargazin . Our data indicate that extracellular layer of AMPA receptors can get trapped in stable , opened-up conformations , especially upon long exposures to glutamate . In contrast , Stargazin limits this conformational flexibility . Thus , under synaptic conditions , where brief glutamate exposures and the presence of auxiliary proteins dominate , extracellular domains of AMPA receptors likely stay compact during gating .
The nearly 100 billion neurons in our brain create a complex and intricate network that can relay information in a fraction of a second . Two neurons can communicate with each other by forming a synapse , a specialised structure where the two cells come into close contact . There , the signalling neuron releases chemicals that the receiving cell captures through dedicated receptors embedded in its membrane . For example , the AMPA receptor is a complex assemblage of different subunits that quickly transmits information by opening and closing to let ions move into the receiving cell . These receptors are some of the fastest to react to the released chemicals , allowing information to be encoded swiftly . In fact , it is increasingly clear that epilepsy and deficits in mental processes can be associated with AMPA receptors having a faulty activity . Yet , it is still unknown how exactly these proteins work . In particular , previous studies have shown that an AMPA receptor can go through dramatic changes in its structure , with the different subunits being able to spread apart widely . However , these experiments had to be conducted when the proteins were isolated from membranes and held in a cocktail of activating or deactivating molecules for hours . It is still unclear whether the results hold when AMPA receptors sit at the membrane while assembled with their partner proteins , like they normally do in the brain . Baranovic and Plested went on to investigate this question by using ‘molecular rulers’ . These tiny molecules have different lengths , and they act as yardsticks: their sticky ends can attach to specific areas in the protein , helping to measure how these regions move relative to each other when the receptors are on or off . A method called patch clamp electrophysiology was used to determine how much the normal activity of the AMPA receptors was hindered by being bound by the molecular rulers . The results showed that AMPA receptors can undergo large structural changes but these movements require time and are much reduced by partner proteins . In the brain , AMPA receptors in synapses probably lack the freedom and opportunity to move so dramatically when neurons are communicating with each other . Ultimately , knowing how these receptors work and move may help grasp the changes in their activity that cause connections between neurons to become defective .