Domain Organization and Function in Gluk2 Subtype Kainate Receptors

Domain organization and function in GluK2 subtype kainate receptors

Utpal Dasa, Janesh Kumara, Mark L. Mayera,1, and Andrew J. R. Plestedb,c,1

aLaboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892; bLeibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; and cNeurocure, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany

Edited by Roger A. Nicoll, University of California, San Francisco, CA, and approved March 30, 2010 (received for review January 21, 2010) Glutamate receptor ion channels (iGluRs) are excitatory neurotrans- and solved structures of the ATD dimer assembly by x-ray diffraction mitter receptors with a unique molecular architecture in which the (10). In both crystal forms the subunits in a dimer assembly show 2-fold extracellular domains assemble as a dimer of dimers. The structure molecular symmetry, with a buried surface area of 1540 Å2 per subunit of individual dimer assemblies has been established previously for almost equally distributed between domains R1 and R2 (10). From both the isolated ligand-binding domain (LBD) and more recently each of these structures a tetrameric assembly composed of AB and for the larger amino terminal domain (ATD). How these dimers A′B′ subunit dimers can be generated by application of crystal sym- pack to form tetrameric assemblies in intact iGluRs has remained metry operations (Fig. 1A). In the resulting N-shaped structure, there controversial. Using recently solved crystal structures for the GluK2 is a global 2-fold axis of symmetry, with contacts between the pair of kainate receptor ATD as a guide, we performed cysteine mutant dimer assemblies mediated exclusively by residues at the lateral edge cross-linking experiments in full-length tetrameric GluK2 to estab- of domain R2. The GluK2 ATD N and C termini, deﬁned by the CA lish how the ATD packs in a dimer of dimers assembly. A similar positions of Thr2 and Glu384 of each subunit, form twisted paral- approach, using a full-length AMPA receptor GluA2 crystal structure lelepipeds with dimensions of 102 × 68 Å and 51 × 63 Å, respectively as a guide, was used to design cysteine mutant cross-links for the (Fig. 1B). The buried surface area of the interdimer interface is 600 Å2 GluK2 LBD dimer of dimers assembly. The formation of cross-linked per subunit and involves 2-fold symmetric contacts between helices tetramers in full-length GluK2 by combinations of ATD and LBD H, I, and K, including a salt bridge between Lys188 and Glu219,

mutants which individually produce only cross-linked dimers sug- ahydrogenbondbetweenLys188andthemainchaincarbonylof NEUROSCIENCE gests that subunits in the ATD and LBD layers swap dimer partners. Ser241, and a potential cation π bond between Lys181 and Tyr240. van Functional studies reveal that cross-linking either the ATD or the der Waals contacts are formed between the side chains of Lys188, LBD inhibits activation of GluK2 and that, in the LBD, cross-links Leu212, Met214, Thr-218, Glu219, Tyr220, and Tyr240 (Fig. 1C). within and between dimers have different effects. These results establish that kainate and AMPA receptors have a conserved ATD Cysteine Mutants Map Dimer Interaction Surfaces in Full-Length extracellular architecture and provide insight into the role of GluK2. To establish whether packing in full-length GluK2 matches individual dimer assemblies in activation of ion channel gating. that seen in the isolated ATD, we generated a series of cysteine mutants and tested these for oligomer formation by Western blot crystal structure | glutamate receptors | ligand gated ion channels analysis under oxidizing conditions. The wild-type GluK2 construct contains 12 native cysteine residues (12), and in control lutamate receptor ion channels (iGluRs) mediate excitatory experiments we observed weak cross-linking which probably would Gtransmission in the mammalian brain (1). Understanding interfere with analysis of the effect of cysteines introduced to test how they transduce the binding energy of glutamate into rapid mechanisms of GluK2 assembly (Fig. 2). Analysis of GluK2 ATD activation and desensitization of ion channel gating requires and LBD crystal structures reveals that the pairs Cys-65 and -316 in structural information. For nicotinic acetylcholine receptors the ATD and Cys-719 and -773 in the LBD form disulfide bonds and related members of the large family of cysteine (Cys) loop and that Cys-91, -199, and -432 are buried in the protein interior receptors (2–4), and for acid-sensing ion channel and P2X (10, 13). Mutation of all of the remaining five cysteine residues to receptors which bind protons and ATP (5, 6), the extracellular their counterparts in the GluA2 AMPA receptor was required to A LBDs exhibit 5- and 3-fold rotational molecular symmetry about eliminate spontaneous cross-linking (Fig. 2 ); the mutations a central axis centered on the ion channel pore. By contrast, made were C540Y; C545V, an RNA editing site in M1 (12); C564S iGluRs are tetrameric assemblies in which the extracellular in the cytoplasmic linker connecting M1 and M2; and C827N and amino terminal domain (ATD) and ligand-binding domain C840Q, two palmitoylation sites in the cytoplasmic C terminus × – (LBD) both assemble as a dimer of dimers (7–10). Surprisingly, (14). Functional assays on the resulting 5 cysteine ( ) mutant Xenopus in the recently solved structure of a full-length AMPA receptor, expressed in oocytes and HEK cells revealed that voltage- the subunit pairs that form the ATD dimer assemblies swap dependent polyamine block, attenuation of desensitization by the partners in the LBD (11). Here we test whether the assembly lectin Con A, and the kinetics and extent of desensitization were not different from wild type. For example, in rapid perfusion principles observed in GluA2 apply also to kainate receptors, −1 experiments, kdes = 129 ± 13 s , and steady-state desensitization identify the interfaces which form the dimer of dimers assembly ± μ in both the ATD and LBD of GluK2, and examine the functional reached 98.4 0.4% in 10 M copper phenanthroline (CuPhen), effects of cysteine mutant disulfide cross-links in the ATD and LBD dimer assemblies. Our results suggest that the LBDs in Author contributions: M.L.M. and A.J.R.P. designed research; U.D., J.K., M.L.M., and A.J.R. a homomeric glutamate receptor tetramer are not functionally P. performed research; U.D., J.K., M.L.M., and A.J.R,P. analyzed data; and M.L.M. and A.J. equivalent and have implications for understanding how het- R.P. wrote the paper. eromeric receptors function in the brain. The authors declare no conflict of interest. Results This article is a PNAS Direct Submission. 1To whom correspondence may be addressed. E-mail: [email protected] or plested@ Crystal Structure of the GluK2 ATD Tetramer Assembly. We pre- fmp-berlin.de. viously crystallized the amino terminal domain of GluK2 in two This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. different forms [Protein Data Bank (PDB) 3H6G and 3H6H] 1073/pnas.1000838107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1000838107 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Fig. 2. GluK2 ATD cross-links created by cysteine mutagenesis. (A) The blots show (left to right) spontaneous oligomerization for wild-type GluK2; the C540Y/C545V/C564S 3× (–) triple mutant; and the4× (–) mutant which contained in addition the exchange C827N. The additional change, C840Q, to produce the 5× (–) mutant yielded a construct which ran as a monomer with the same mo- Fig. 1. Crystal structure of the GluK2 ATD tetramer. (A) Molecular surface bility as wild-type GluK2 under reducing conditions. (B) The L151C mutation in viewed from the top (Left) and after rotation by 90° (Right) with the four the AB subunit ATD dimer interface produced spontaneous dimer formation subunits colored individually. (B) Ribbon diagram showing how the GluK2 (control, Con), which was not enhanced by incubation with CuPhen (oxidizing ATD AB and CD subunit dimers pack via the lateral edges of domain R2 to condition, Ox) and which was reversed by incubation with 10 mM BME (re- form a tetramer; the CA atoms of Thr2 and Glu384, which define the N and C ducing condition, Red). (C) The G215C mutant in the BD subunit interdimer termini of the ATD in each subunit, are indicated by colored spheres con- interface also produced spontaneous dimer formation which was not enhanced nected by dashed lines; black dumbbells indicate the CA positions of L151 in by incubation with CuPhen. (D) The M214C/Y240C double mutant, designed to the AB and CD dimers. (C) Stereo view of the interaction surface connecting cross-link helices I and K in the BD subunit tetramer interface, also produced subunits B and D in the GluK2 ATD tetramer. Side chains that mediate dimer formation. (E) The L151C/ G215C double mutant showed bands for both intersubunit contacts are represented as sticks; orange spheres indicate the dimers and tetramers. (F) Outside-out patch responses for GluK2 5× (–)had CA positions of Gly215. The view matches that in the right panel in A. kinetics similar to wild-type GluK2; in oxidizing conditions the current in CuPhen −1 (red curve; monoexponential fit, kdes =131s ) was 88% of the peak current in −1 DTT (blue curve; kdes =117s ), but on average, peak currents in CuPhen were − with similar values of 132 ± 16 s 1 and 97.2 ± 1.2% in 10 mM DTT 101 ± 14% of the amplitude of those in DTT. (G) In reducing conditions; 10 mM n F glutamate activated rapidly desensitizing currents for the L151C mutant (blue ( = 6) (Fig. 2 ). Both parameters are similar to previously −1 −1 fitted curve; kdes = 100 s ). The peak amplitude was inhibited in oxidizing reported values of 165 s and 99.1% for wild-type GluK2 (8, 15), fi −1 μ ± × – conditions (red tted curve; kdes =102s ; current in 10 M CuPhen was 49 7% although on average the peak current for the 5 cysteine ( ) was of that in 10 mM DTT, P = 3%, n = 6 patches). The inhibition was readily re- about 5- to 10-fold smaller than for wild type. −1 versible (green fitted curve; kdes =89s ). (H) The G215C mutant also was re- −1 Using nomenclature developed for the full-length AMPA re- versibly inhibited in oxidizing conditions (red fitted curve; kdes =63s ); control A′ B′ −1 ceptor, we will refer to the and peptide chains in the GluK2 and wash responses in DTT were similar (blue and green curves; kdes =61s and −1 fi ATD tetramer as subunits “D” and “C.” In the isolated GluK2 kdes =60s , respectively). The inhibition was statistically signi cant; peak cur- ATD crystal structure, AB or CD subunit dimer assemblies are rent in CuPhen was 55 ± 8% of the DTT current (P < 0.05, n = 6). (I) L151C-G215C double mutants were more strongly inhibited in oxidizing conditions (red fitted stabilized by a hydrophobic surface in domain R2 in which the −1 curve; kdes =74s ; peak amplitude in CuPhen was 35 ± 4% of that in DTT, n =4). CA atoms of Leu151 in the two subunits in a dimer pair are − The inhibition was fully reversible (green fitted curve; k =74s 1). separated by 6.8 Å (Fig. 1B). Consistent with ideal geometry for des disulfide bond formation revealed by modeling cysteine mutant rotamers, the L151C mutant formed cross-linked dimers spontaneously, and the extent of dimerization was not increased by formed by contacts between the B and D subunits in full-length application of 10 μM CuPhen (Fig. 2B). In the GluK2 ATD GluK2 was obtained using the double mutant M214C/Y240C tetramer, the interdimer interface is formed by helix I of subunit (Fig. 2D), which links the top of helix H in subunit B with the B, which projects into a pocket formed by helices I and K of base of helix K in subunit D (Fig. 1B). When the L151C and subunit D in the adjacent dimer, and vice versa. Introduction of G215C mutations were combined to cross-link simultaneously a cysteine at the base of helix I produced spontaneous GluK2 the AB, CD, and BD subunit dimer interfaces, Western blots cross-linking for the G215C mutant, and the extent of di- revealed the formation of tetramers as well as dimers (Fig. 2E). merization was not increased by application of 10 μM CuPhen In all cases, samples reduced by incubation with 10 mM beta- (Fig. 2C). Further evidence for a dimer of dimers assembly mercaptoethanol (BME) migrated as monomers.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1000838107 Das et al. Downloaded by guest on September 27, 2021 Functional Effects of ATD Cross-Links. We used outside-out patch recording with rapid application of 10 mM glutamate to test for functional effects of ATD cysteine mutant cross-links and fo- cused on the L151C and G215C mutants identiﬁed by Western blot analysis (Fig. 2). We applied glutamate every 10 s and al- ternated every 5–10 responses between reducing conditions with 10 mM DTT and test responses under oxidizing conditions with 10 μM CuPhen (16). Control experiments revealed that the GluK2 5× (–) construct had activation and desensitization kinetics similar to wild-type GluK2 and that responses to glutamate were not sensitive to redox conditions (Fig. 2F). The G215C mutant also showed rapid activation and nearly complete desensitization (Fig. 2G), but responses to glutamate showed reversible inhibition by CuPhen. After oxidation the peak current was 55 ± 8% (P < 0.05, n = 7 patches) of that in DTT (kdes − − 93 ± 10 s 1 in DTT and 107 ± 14 s 1 in CuPhen), slightly slower − than the value of 165 s 1 for wild-type GluK2 (16). The L151C mutant also produced redox-sensitive responses (Fig. 2H), such that glutamate-activated peak currents in CuPhen were reduced in amplitude to 49 ± 7% of those in DTT (P < 0.05, n =5 patches). Again, desensitization was apparently unaffected (kdes 103 ± 15 s−1 in DTT and 114 ± 21 s−1 in CuPhen). When the G215C and L151C mutants were combined to produce a covalently linked tetrameric ATD assembly, the de- gree of inhibition in CuPhen (peak current, 35 ± 4% of that in 10 mM DTT) was greater than for the single ATD mutants (Fig. 2I). In fact, the extent of inhibition was similar to the

product for the single mutants, suggesting that the accumulated NEUROSCIENCE cross-links independently limit the ability of glutamate to activate the channel. The rate of desensitization for the G215C/ L151C double mutant was slowed compared with both wild-type −1 GluK2 and the individual point mutants [kdes 73 ± 19 s in DTT − and 86 ± 18 s 1 in CuPhen (n = 5)] but remained insensitive to the redox condition.

GluK2 Ligand-Binding Tetramer Assembly. Earlier structural work had established that the GluK1 and GluK2 LBDs assemble as homodimers (17, 18) that can be stabilized in their active conformation by protein engineering (19, 20) and by domain 1 cysteine mutants (8). Insight into how the LBD dimers pack to form a kainate receptor tetramer remained elusive, and low-resolution single-particle EM image analysis for AMPA receptors gave contradictory results (21–23). The crystal structure of a full- length AMPA receptor now has provided the necessary higher- resolution template to test assembly mechanisms in kainate Fig. 3. GluK2 LBD cross-links created by cysteine mutagenesis. (A) Molecular receptors (11). We tested for formation of cross-links at the surface of the GluA2 LBD tetramer (11) with each subunit colored using the interdimer surface of the tetrameric LBD assembly by in- same scheme as for the GluK2 ATD tetramer shown in Fig. 1. (B) Ribbon troducing cysteine mutants at two different locations in domain 2 diagram for a homology model of the GluK2 LBD tetramer showing cysteine of the GluK2 LBD. Based on subunit terminology derived from mutants designed to cross the A and C subunits and the A and B subunits in the full-length AMPA receptor structure, mutants proximal to a tetramer assembly. (C) Western blot analysis for cross-links connecting the the global 2-fold axis of LBD symmetry will link the A and C GluK2 LBD tetramer interface. The K667C mutation, which cross-links the subunits, whereas distal mutants cross-link the A and B or C and loop connecting helices F and G in the AC subunit interdimer interface, A produced spontaneous dimer formation, which was slightly enhanced by D subunits in adjacent dimer assemblies (Fig. 3 ). In addition we incubation with CuPhen (Ox) and reversed by 10 mM BME (Red). The S669C combined mutations in the LBD with those in the ATD to test mutation at the N terminus of helix G also produced cross-linking of subunits whether the subunit cross-over observed in the full-length GluA2 in the AC subunit interdimer interface. The A676C/G770C double mutant, crystal structure also occurs in kainate receptors. which cross-links subunits A and B in the LBD tetramer, produced weak The ﬁrst two mutations tested, K667C and S669C, cross-link spontaneous dimer formation, which was strongly enhanced by incubation the N terminus of helix G in subunit A with its proximal dimer with CuPhen. A similar effect was observed for the S680C/N771C double partner in subunit C (Fig. 3B) and are equivalent to K663 and mutant, which also cross-links subunits A and B. (D) The double mutant I664 in the AMPA receptor GluA2 subunit (11, 16). The second L151C/K667C, which cross-links both the AB and CD subunit ATD dimer set of mutants tested is distal to the global axis of symmetry and interfaces and the AC subunit dimer interface in the LBD, produced spontaneous dimer and tetramer formation, which was reversed by 10 mM BME; cross-links the C terminus of helix G in subunit A with the C the cartoon shows the intersubunit connections in this mutant. terminus of helix K in subunit B (and potentially with their symmetry equivalents in subunits C and D) (Fig. 3B). The K667C and S669C mutants both formed disulﬁde cross-linked dimers and N771C which link helices G and K with their distal dimer spontaneously, and the extent of dimerization was not increased partners, but, in contrast to results for proximal mutants, the by application of 10 μM CuPhen (Fig. 3C). Cross-links also could formation of cross-links was very strongly stimulated by 10 μM be formed by the pair A676C and G770C and by the pair S680C CuPhen (Fig. 3C).

Das et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 We next investigated if the symmetry switch between the ATD and LBD “layers” seen in the AMPA receptor GluA2 crystal structure is also a feature of kainate receptor architecture. In GluA2, the dimer pairs in the ATD layer are formed by subunits AB and CD, whereas the LBD dimers are formed by subunits AD and BC (11). Thus we combined single-interdimer mutations in the ATD and LBD layers of GluK2 in an attempt to cross-link tetrameric assemblies (SI Materials and Methods). The GluK2 L151C mutant, which cross-links the A and B or C and D subunits in the ATD, combined with the K667C mutant, which cross-links the A and C subunits in the LBD, produced both dimer and tetramer formation (Fig. 3D), consistent with a cross- over of the subunits forming dimer assemblies in the ATD and LBD extracellular layers of a GluK2 receptor assembly (Fig. 3D).

Subunit Nonequivalence in the Ligand-Binding Domain Tetramer. The LBD cysteine mutants used for our experiments were designed to produce intermolecular cross-links based on the crystal structure of an AMPA receptor trapped in the resting state by a competitive antagonist (11). These mutants would be expected to interfere with movements produced by agonist binding and thus limit subsequent receptor activation or desensitization. In contrast to the similar effects observed for intradimer and interdimer ATD cross-links, we observed distinct patterns of inhibition produced by cross-links between different pairs of subunits in the GluK2 LBD tetrameric assembly, revealing a functional distinction between subunits located at proximal and distal positions relative to the overall 2-fold axis of the receptor. In oxidizing conditions, disulfide bonds that formed between the K667C or S669C mutants in diagonally opposed A and C subunits, proximal to the global axis of symmetry, led to a partial but readily reversible inhibition of the glutamate-activated current (Fig. 4 A and B). On average, compared with responses recorded in DTT, glutamate-activated peak currents were reduced in amplitude to 35 ± 8% (P < 0.05, n = 6) for K667C and 56 ± 11% (P < 0.02, n = 7) for S669C. These diagonally cross- linked mutants also showed reduced desensitization, so that, when oxidized, the steady-state current was larger than control (Fig. 5). This effect was most profound in the S669C mutant for which the steady-state current was 7 ± 2% of the peak in DTT, Fig. 4. Effects of LBD cross-links on GluK2 receptor function. (A) The K667C whereas in CuPhen the steady-state current increased about 4- mutant showed partial inhibition by oxidation. In this patch, the peak current amplitude was reduced from 153 pA to 30 pA, and the rate of desensitization fold, so that the ratio of steady-state to peak current was 26 ± 5% −1 −1 P n was similar in DTT and CuPhen (kdes =188s and 152 s , blue and red curves, ( =0.016, = 7 patches). Linked to this effect were reproducible respectively); the extent of desensitization was reduced in CuPhen, so that rel- −1 changes in the rate of desensitization: kdes 140 ± 16 s in DTT ative to the peak steady-state currents were 5.3 ± 0.7-fold larger than in DTT. −1 −1 and 249 ± 17 s in CuPhen for K667C; kdes 66 ± 10 s in DTT K667C responses frequently showed rundown, as illustrated by incomplete re- −1 −1 and 96 ± 24 s in CuPhen for S669C (Fig. 5). Overall, the be- covery of the peak amplitude upon return to DTT (green curve; kdes =187s ). (B) havior of receptors harboring diagonal cross-links was similar to The S669C mutant exhibited fast desensitization in reducing conditions (blue −1 curve; kdes 72 s ) that was slightly, but significantly, accelerated in oxidizing that observed for the I664C mutant in GluA2 (16) and resembles −1 responses produced by some partial agonists at kainate and conditions (red curve; kdes 100 s ). As for the K667C mutant, the response was AMPA receptors. only partially (39% in this example) inhibited by oxidation. Desensitization was blocked substantially, with the steady-state current increasing to 19% of the Interdimer cross-links distal to the global 2-fold axis of pseu- peak in oxidizing conditions, compared with 2% in 10 mM DTT. (C) Glutamate- dosymmetry at the AB or CD interfaces caused a more profound activated currents for the S680C-N771C mutant were much more strongly inhibition of responses to glutamate but no increase in steady- inhibited in CuPhen (peak amplitude 9% in this example); the inhibition was state current (Fig. 4C). Compared with responses in DTT, glu- rapidly and readily reversible, as shown by blue- and green-colored exponential tamate-evoked currents were 6-fold smaller in CuPhen for fits for control and recovery responses, respectively. A676C-G770C (peak amplitude 16 ± 4%, n = 6) and 10-fold smaller in CuPhen for S680C-N771C (peak amplitude 8 ± 2%, n = 6). The large inhibition of responses to glutamate under linking experiments (8, 25), and site-directed mutagenesis (19, 26) oxidizing conditions unfortunately limited our ability to measure reveal that, despite their common fold, the LBDs of glutamate changes in the rate of onset of desensitization accurately, but receptors exhibit striking functional variation and that this vari- when accurate measurement was possible, differences between ation often is underpinned by exquisitely subtle structural changes these rates of responses in CuPhen and DTT were not statisti- (15, 27). The isolated ATDs of GluK2 and GluA2 also have cally significant (Table S1). similar structures, but much less work has been performed on this domain (9, 10, 28). We find that the interdimer interface in the Discussion GluK2 ATD tetramer is more tightly packed than that in AMPA Our results establish that the tetrameric assembly of GluK2 kai- receptors, with a buried surface of 600 Å2, whereas for the cor- nate receptors closely resembles that of AMPA receptors. How- responding GluA2 structure (PDB 3H5V) the buried surface is ever, high-resolution crystal structures (24), functional cross- only 400 Å2 and decreases to only 330 Å2 in full-length GluA2

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1000838107 Das et al. Downloaded by guest on September 27, 2021 desensitization based on the full-length GluA2 crystal structure, which predicts large changes in the distance within and between ATD dimers (11), the ATD cross-links reported here do not alter the extent or rates of onset and recovery from desensitization (Fig. S1). Our results show that although crystal- lographic studies provide a rich framework for understanding glutamate receptor function, many basic details about the role of the ATD in gating mechanisms remain to be determined. By contrast, a large body of work on the LBD of kainate and AMPA receptors provides a rational framework for explaining the diverse effects of cysteine mutants. For both GluK2 and GluA2, the domain 1 Y490C/L752C mutants stabilize the active conformation of the LBD dimer assembly and block desensitization (8), whereas the domain 2 G725C and S729C mutants, which trap GluA2 in the desensitized state, abolish responses to glutamate (16, 25). Here we report two different classes of domain 2 cysteine mutants that inhibit responses to glutamate. The GluA2 full-length crystal structure reveals that these mutants map to a previously unknown interdimer interface formed at the base of helix G in the antagonist bound resting state. The inhibitory effect of these mutants is consistent with the intuitive notion that contacts mediated by domain 2 interfaces in the resting state are likely to be broken upon receptor activation (11). Surprisingly, the K667C and S669C mutations that link the proximal A and C subunits in the LBD tetramer produce partial inhibition of the glutamate-activated peak current but also re- duce desensitization, leading to an increase in steady-state cur-

rent, whereas the A676C/G770C and S680C/N771C mutations NEUROSCIENCE that link the distal subunits produce only a strong inhibition of activation. What leads to partial inhibition and reduced desensitization observed for the K667C and S669C mutants? A clue comes from studies on native AMPA and kainate receptors for which partial agonists such as the 5-substituted willardiines and kainic acid exhibit a negative correlation between efficacy and the extent of desensitization, mirroring responses for the GluK2 S669C mutant (33–35). Crystal structures show that these partial agonists produce less LBD closure than glutamate. Interdimer cross-links Fig. 5. Bar plots illustrating kinetic effects of cross-links in the LBD. (A) Oxidizing conditions producing cross-links between AC and AB subunits give rise to distinct at S669C and K667C also would be expected to restrict domain patterns of current inhibition. The 5× (–) construct was not inhibited by oxidation. closure for the LBD of each subunit in a dimer pair, suggesting Intermediate inhibition was observed for the K667C and S669C mutants, and a related mechanism of action. Alternatively, the smaller peak much stronger reduction of the current was seen for the S680C-N771C and the current and reduced desensitization seen for the S669C mutant A676C-G770C double mutants; number of patches indicated above bars. (B)Ox- could result from changes in gating kinetics and possibly agonist idation produced small increases in desensitization rate for all mutants, but the affinity. However, we excluded this possibility because the rates effect was statistically significant only for the K667C and S669C mutants (P =0.03 of both deactivation and recovery from desensitization are not for each, randomization test). Only a subset of responses in CuPhen for S680C- greatly accelerated in the S669C mutant (Fig. S1). N771C (three of six patches) and A676C-G770C (four of six patches) were large Single-channel recording indicates that individual iGluR sub- enough to measure the desensitization rate accurately. (C) The steady-state current was strongly and significantly increased in amplitude for K667C and units can activate sequentially following the binding of glutamate S669C but not for the AB subunit cross-linking mutants. *, P, < 0.05. (36), but at present it is not known if the four subunits in a tetramer produce equal activation of ion channel gating. In- complete inhibition of glutamate responses by the K667C and (PDB 3KG2). This difference also extends to the more extensive S669C mutants might occur because only the subunits proximal ATD dimer interface between the AB or CD subunits, which has – 2 to the pseudo 2-fold axis of symmetry are cross-linked, leaving a buried surface area of 1,657 and 1,540 Å in the two GluK2 the distal subunits free to close when glutamate binds. Re- crystal forms (10), whereas for full-length GluA2 and the GluA2 2 striction of the movement of distal subunits by the A676C/ isolated ATD the buried area is only 1,334 Å (9, 11, 28). G770C and S680C/N771C mutants inhibited responses to glu- Very little is known about the role, if any, of the ATD in either tamate more profoundly, consistent with either a dominant role AMPA or kainate receptor gating. Allosteric signaling by the in driving channel opening or formation of cross-links for both – ATD is well established in NMDA receptors (29 31) but not in the AD and BC dimer pairs. In a symmetrical dimer of dimers non-NMDA receptors, with the possible exception of kainate assembly, cysteines in helix K of both distal subunits should form receptor modulation by polyamines and protons (32). Our results cross-links with their subunit proximal partners. However, the reveal that cross-links both within and between ATD dimer GluA2 full-length structure has local asymmetry in the LBD, and assemblies, produced by cysteine mutants that form redox-sen- only one pair of subunits is within disulfide-binding distance (11). sitive disulfide bonds, modestly inhibit activation by glutamate. Our data do not provide information about the conservation of The mechanism underlying this effect is uncertain, but it is this asymmetry in kainate receptors or about whether in possible that the formation of cross-links causes subtle changes a membrane-embedded receptor it is possible for simultaneous in the orientation of the ATD, which in turn propagates to the cross-linking to occur at both sites, but the small glutamate re- LBD. Surprisingly, in view of models for glutamate receptor sponse observed in oxidizing conditions (16 ± 4% of that in DTT

Das et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 for A676C/G770C) is consistent with the asymmetry observed in Materials and Methods the crystal structure. Our results thus raise provocative questions Molecular Biology and Biochemistry. For thisstudy weused the pRK5 expression about whether ion channel gating triggered by activation of the vector encoding rat GluK2 (previously called “GluR6”) with the three RNA proximal and distal LBD dimer pairs is functionally equivalent. editing sites encoding VCQ and with a C-terminal His tag. Mutants were The stronger inhibition observed for the A676C/G770C and generated using overlap PCR and confirmed by double-stranded DNA se- S680C/N771C distal subunit mutants suggests that these pairs quencing of the amplified region. The numbering refers to the mature poly- could provide the majority of the gating force to open the peptide chain following cleavage of the signal peptide (10). HEK-293T cells channel, but further work is required to test this possibility. grown in T25 flasks were transfected using polyethyleneimine as described In conclusion, it seems probable that there is a hierarchy of previously (10). Following solubilization with DDM, SDS/PAGE with 4–12% intersubunit interfaces in AMPA and kainate receptors. In the gels, and transfer to PVDF, blots were probed using anti GluK2/3 clone NL9 resting state D2 interfaces formed between proximal and distal antibody (Upstate) and developed using the Amersham ECL Western blotting subunits in the LBD are likely to be weak, and we propose that this detection kit (GE Healthcare) and x-ray film (SI Materials and Methods). weakness is essential for efficient receptor activation, because movement of the D2 domains away from the central axis of the Electrophysiology. Wild-type and mutant GluK2 were expressed by transient iGluR tetramer drives ion channel opening. In contrast, the D1 expression in HEK-293 cells for outside-out patch recording with fast solution LBD interface is strong enough to remain intact during receptor exchange as previously described (16). Typical 10–90% solution exchange activation but breaks when the receptor desensitizes. Within the times were 300 μs, as measured at the open tip of the patch pipette. Data ATD, the local dimer interface, which underlies the formation of were fit in Axograph and plotted in Kaleidagraph (Synergy Software). Sta- ATD homodimers with micromolar or higher affinity (9, 10, 28), is tistical significance was assessed using a two-tailed randomization test using likely to be the most stable assembly in the extracellular segment, the program RANTEST (kindly provided by Remigijus Lape and David Colqu- consistent with its proposed role in assembly (37, 38). By contrast, houn, University College London). Additional details are given in SI Materials the small footprint of the ATD dimer–dimer interface suggests and Methods. a much weaker interface. In summary, our results establish the presence of specific, small interfaces in the extracellular domains ACKNOWLEDGMENTS. We thank Carla Glasser for preparing cDNAs; Marcus of kainate receptors and indicate that their arrangement is likely to Wietstruk and Dr. Anna Carbone for assistance with cell culture; Dr. C. be the same as that in AMPA receptors. Movement of these Rosenmund for the loan of a piezoelectric stack; Dr. E. Gouaux for sharing fi GluA2 coordinates before publication, and the National Institute of interfaces has signi cant roles in receptor activation and de- Neurological Disorders and Stroke DNA sequencing facility. This work was sensitization. Further work using a combination of approaches will fi supported by the intramural research program of the National Institute of be required to establish the extent of subunit-speci c domain Child Health and Human Development, National Institutes of Health, movements in iGluRs and how this extent varies during the pro- Department of Health and Human Services (M.L.M.) and the Neurocure cesses of activation and desensitization. Cluster of Excellence, Deutsche Forschungsgemeinschaft (A.J.R.P.).

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