Conformational restriction blocks glutamate receptor desensitization

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1 Conformtionl restriction locks glutmte receptor desensitiztion Mtthew C Weston 1, Peter Schuck 2, Alokesh Ghosl 3, Christin Rosenmund 1 & Mrk L Myer 3 Desensitiztion is universl feture of lignd-gted ion chnnels. Using the crystl structure of the GluR2 L483Y mutnt chnnel s guide, we ttempted to uild non-desensitizing kinte-sutype glutmte receptors. Success ws chieved for GluR5, GluR6 nd GluR7 with intermoleculr disulfide cross-links ut not y engineering the dimer interfce. Crystllogrphic nlysis of the dimer reveled relxtion from the ctive conformtion, which functionl studies revel is not sufficient to trigger desensitiztion. The equivlent non-desensitizing cross-linked GluR2 mutnt retined wek sensitivity to positive llosteric modultor, which hd no effect on GluR2 L483Y. These results estlish tht the ctive conformtion of AMPA nd kinte receptors is conserved nd further show tht their desensitiztion requires dimer rerrngements, tht sutle structurl differences ccount for their diverse functionl properties nd tht the lignd-inding core dimer is powerful regultor of ion-chnnel ctivity. Lignd-gted ion chnnels t chemicl synpses re ctivted y smll diffusile neurotrnsmitters such s glutmte, cetylcholine, glycine nd ATP 1. Despite their moleculr diversity, these receptors shre the common properties of rpid ctivtion of ion-chnnel gting in response to the inding of gonists, followed y desensitiztion in the continued presence of neurotrnsmitter. Desensitiztion, process tht decouples gonist inding from receptor ctivtion, modultes synptic responses during high-frequency ctivity 2,3 nd lso prevents cell deth resulting from unregulted ion flux nd the collpse of trnsmemrne electrochemicl grdients during excessive receptor ctivtion 4,5. The occurrence of desensitiztion in structurlly diverse receptor fmilies suggests strong evolutionry pressure for its conservtion, ut for the mjority of lignd-gted ion chnnels, the moleculr mechnisms hve yet to e elucidted. Understnding of the mechnism of desensitiztion is most dvnced for AMPA-sutype glutmte receptors 6,7. The identifiction of llosteric modultors of desensitiztion 8,9 nd AMPA receptor mutnts with spectrum of desensitiztion rtes 6,7,10, comined with iochemicl nd crystllogrphic techniques 6,11, hs een key in contriuting to this dvnce. Drugs tht ttenute AMPA receptor desensitiztion might lso e memory nd cognition enhncers 12. In contrst, much less is known out the moleculr mechnisms underlying desensitiztion of the two other mjor fmilies of glutmte receptors, the kinte nd NMDA receptors. Although their gene fmilies show only 38% 41% mino cid sequence identity 13, AMPA nd kinte receptors re usully regrded s close cousins. It is thus noteworthy tht ttempts to generte muttions tht lock kinte receptor desensitiztion hve not succeeded 10,14,15. The resons for this re unknown, nd this rises the possiility tht AMPA nd kinte receptors hve distinct gting mechnisms. Indeed, severl recent studies highlight differences in the functionl properties of AMPA nd kinte receptors 14,16 18, the underlying mechnisms of which re not understood. Here we use comintion of crystllogrphic, iochemicl nd electrophysiologicl nlyses to ddress the mechnism of kinte receptor desensitiztion nd descrie for the first time fmily of non-desensitizing mutnts from the Rttus norvegicus GluR5, GluR6 nd GluR7 fmily of kinte receptors. Our pproch used disulfide ond cross-links s form of conformtionl restrint preventing ccess to the desensitized conformtion. In contrst to prior work, this pproch locks desensitiztion of oth AMPA nd kinte receptor sutypes. Our results suggest tht the ctive conformtion of AMPA nd kinte receptors is conserved, tht their mechnism of desensitiztion requires reorgniztion of lignd-inding core dimers nd tht sutle structurl differences ccount for their diverse functionl properties. RESULTS Structurl similrity of AMPA nd kinte receptors The ccepted model for gting of glutmte receptor ion chnnels ssumes dimer-of-dimers ssemly. The lignd-inding site of ech suunit is formed y two-domin clm shell like structure, the closure of which triggers ion-chnnel gting (Fig. 1).The ctive stte of the receptor is stilized y ck-to-ck ssemly in which the individul protomers in dimer pir re stilized y extensive 1 Deprtments of Neuroscience nd Moleculr nd Humn Genetics, Bylor College of Medicine, Houston, Texs 77030, USA. 2 Protein Biophysics Resource, Division of Bioengineering nd Physicl Science, Office of Reserch Services, nd 3 Lortory of Cellulr nd Moleculr Neurophysiology, Porter Neuroscience Reserch Center, Ntionl Institute of Child Helth nd Humn Development, US Ntionl Institutes of Helth, Deprtment of Helth nd Humn Services, Bethesd, Mrylnd 20892, USA. Correspondence should e ddressed to M.L.M. (myerm@mil.nih.gov). Received 27 June; ccepted 31 Octoer; pulished online 19 Novemer 2006; doi: /nsm VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

2 Rest Desensitized 25 Open 10 mm glutmte WT GluR2 200 pa 15 ms GluR2 L483Y 25 contcts formed y the domin 1 surfce of ech protomer This ssemly trnsltes gonist-triggered domin closure into n increse in seprtion of the juxt-memrne linkers tht led to the lipidemedded ion-chnnel domins 19,23. There is sustntil evidence tht AMPA receptor desensitiztion egins with relxtion of the dimer interfce, ut it is not known whether this mechnism pplies to other glutmte receptor sutypes. Some of the most compelling evidence for this model comes from iochemicl, crystllogrphic nd functionl studies with the non-desensitizing GluR2 L483Y mutnt 10 nd with llosteric modultors of desensitiztion, which ct y common mechnism: stiliztion of the ctive conformtion of the dimer ssemly 6,11, preventing the uncoupling tht leds to desensitiztion nd ionchnnel closure (Fig. 1). In the GluR2 L483Y dimer, the romtic side chin projects into slot on the domin 1 surfce of its prtner suunit 6,incresingthe ffinity for dimer formtion more thn fold. Lest-squres superpositions of dimer crystl structures for kinte receptors on the GluR2 L483Y mutnt crystl structure 6 gve r.m.s. devitions of 1.02 nd 0.94 Å for C toms in domin 1 of wild-type GluR5 (ref. 23) nd GluR6 (ref. 21), respectively, indicting very similr structures (Fig. 1). The uried surfce re is comprle for the three receptor sutypes, with vlues of 1,080 Å 2 per suunit for GluR2 L483Y, 1,078 1,080 Å 2 for GluR5 nd 1,367 1,431 Å 2 for GluR6. If desensitiztion proceeds y similr mechnisms in kinte nd AMPA receptors, it should e possile generte high-ffinity inding site for the ntive tyrosine side chin of kinte receptors y protein engineering nd therey recrete the non-desensitizing phenotype of the GluR2 L483Y mutnt. R2 L483Y WTR5 WTR6 Figure 1 Structurl sis for desensitiztion of glutmte receptor ion chnnels. () Crtoon of n AMPA receptor dimer during trnsitions from the resting, lignd-free stte to the glutmte-ound open stte nd the desensitized stte. Chnnel opening results from domin closure; desensitiztion is triggered y seprtion of domin 1 dimer contcts, llowing the pir of suunits to rotte nd close the ion chnnel. Chrt shows superimposed responses to 10 mm glutmte recorded from outsideout ptches from HEK cells trnsfected with wild-type GluR2 nd the nondesensitizing L483Y mutnt; the onset of desensitiztion for the wild-type response is fit with single exponentil of time constnt 6 ms, shown in red. () C trces for dimer crystl structures of the GluR2 L483Y mutnt complex with AMPA (green, PDB 1LB8), the wild-type GluR5 complex with glutmte (red, PDB 2F36) nd the wild-type GluR6 complex with domote (lue, PDB 1YAE). The structures were superimposed y the lest-squres method using domin 1 coordintes; view t top is from the N terminus, looking down onto the plne of the memrne; view t ottom is rotted y 901. Owing to differences in domin closure, the coordintes for domin 2 do not superimpose nd re drwn with trnsprent shding. 90 Protein engineering does not lock desensitiztion Crystl structures for wild-type kinte receptor GluR5 nd GluR6 lignd-inding core dimers revel conformtionl flexiility of the ntive tyrosine side chin. In GluR5 glutmte complex, the Tyr490 side chin projects ck towrd its prent suunit 23 (Fig. 2). In GluR6 complex with the prtil gonist domoic cid, Tyr490 dopts different conformtion nd projects into the dimer interfce, s in GluR2 L483Y 21. Amino cid sequence lignments revel conserved mino cids in helices D nd J tht differ etween AMPA nd kinte receptors (Fig. 2) nd tht form high-ffinity inding site for the GluR2 L483Y mutted side chin tht is sent in kinte receptors. In helix J, the exchnge of Ile765 for the leucine present in AMPA receptors produces steric clsh etween the isoleucine C tom nd the Tyr506 side chin when it is rotted into the conformtion found in the GluR2 L483Y mutnt; this d contct cn e removed y sustitution of leucine, which hs n unrnched side chin. The exchnge of Gln769 for the lysine present in GluR2 removes ction p contct with the tyrosine side chin, which is mjor contriutor to the stility of dimer formtion y the GluR2 L483Y mutnt 6. The replcement of glutmine t the se of helix J y the glutmte found in kinte receptors introduces potentil inding site for n lterntive conformtion of the lysine side chin, which could disrupt ction p formtion with L483Y or its equivlent in kinte receptors. Finlly, in helix D, the exchnge of Lys510 for the glutmte found in AMPA receptors introduces potentilly competing intrmoleculr ction p contct tht could pull the tyrosine side chin wy from helix J. Introducing these four conserved AMPA receptor residues from helices D nd J into kinte receptors should recrete the inding site for the ntive tyrosine side chin nd thus crete kinte receptors showing high-ffinity dimer formtion nd non-desensitizing phenotype. We therefore creted GluR5 nd GluR6 ELKQ mutnt lignd-inding cores. To our surprise, sedimenttion equilirium nlysis reveled no differences in the tendency of wild-type GluR5 nd the GluR5 ELKQ mutnt to form dimers (Fig. 2c,d). When the dt from these two constructs were fit with monomerdimer model, sttisticl nlysis gve lower limits for the K d of 6 nd 8 mm, respectively. Similr results were otined for GluR6 nd the GluR6 ELKQ mutnt (dt not shown). Consistent with this, electrophysiologicl studies reveled tht the GluR6 ELKQ mutnt remined strongly desensitizing (Fig. 2e), with rte of onset of desensitiztion (k des )of108±6.9s 1 (n ¼ 11), only two-fold slower thn tht of wild-type GluR6 (k des ¼ 232 ± 8.1 s 1, n ¼ 12). Also similr were the respective extents of desensitiztion t equilirium, 99.1 ± 0.33% nd 99.1 ± 0.16%, nd rtes of recovery from desensitiztion, 0.20 ± 0.01 s 1 nd 0.35 ± 0.01 s 1, for the ELKQ mutnt nd wild-type GluR6. We then crystllized the GluR6 ELKQ mutnt lignd-inding core nd solved its structure y moleculr replcement t 1.96-Å resolution. There were three molecules in the symmetric unit, two of which formed two-fold relted dimer (Fig. 2f). Lest-squres superposition of this dimer on the crystl structure of the non-desensitizing GluR2 L483Y mutnt gve n r.m.s. devition of 0.68 Å for domin 1 C toms, indicting nerly identicl structures. Indeed, in the GluR6 ELKQ mutnt, the Tyr490 side chin hs rotted y 1071 compred with the wild-type GluR5 structure, nd it is sndwiched etween the I749L nd Q753K mutted side chins in n identicl conformtion to tht found in the GluR2 L483Y mutnt crystl structure. Although the ELKQ mutnt hs the intended structure, the dditionl inding energy conferred y interction of the ntive tyrosine side chin in helix D with the mutted leucine nd lysine side chins in NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER

3 c A 250 e Glu773 WT GluR2 WT GluR6 WT GluR5 Tyr Rdius (cm) GluR2 L483Y 0.2 na 0.2 na 0.2 na GluR6 ELQK 0.2 na 100 ms helix J must e compensted y dditionl structurl differences etween AMPA nd kinte receptors tht occur elsewhere in the molecule. Becuse the lignd-inding cores of GluR2 nd GluR6 shre only 50% mino cid identity, sustntil work will e required to mp the loction of these sites, especilly when it is considered tht residues remote from the dimer interfce cn lso influence desensitiztion 14. Dimers creted y disulfide ond cross-links We next ttempted to lock the lignd-inding cores of GluR5 nd GluR6 in their ctive conformtion using intermoleculr disulfide ond cross-links. Comprison of the dimer crystl structures for wildtype GluR5 nd GluR6, nd the GluR6 ELKQ mutnt, with tht of the non-desensitizing GluR2 L483Y mutnt (Figs. 1 nd 2) indicted tht unstrined disulfide onds could e introduced y cysteine sustitutions t Tyr506 nd Leu768 in GluR5, Tyr490 nd Leu754 in GluR6, nd His492 nd Leu753 in GluR7 (ref. 24). The lignd-inding cores for GluR5 nd GluR6 contining these cysteine pirs were expressed s solule proteins in Escherichi coli. Contrry to expecttions, the mutnt lignd-inding core proteins purified s monomers; however, dimers formed spontneously fter overnight incution t ph 8.0 (Fig. 3) nd could e isolted from other species y gel-permetion chromtogrphy for susequent equilirium sedimenttion nlysis nd crystlliztion. To determine whether these disulfide ond cross-links would support dimer formtion in full-length kinte receptors, we prepred memrne frctions from Xenopus levis oocytes injected with messenger RNA encoding GluR6 construct contining the sme cysteine d f D * GluR7 486 GluR6 484 GluR5 500 GluRD 478 GluRC 479 GluRB 477 GluRA 473 GluR7 743 GluR6 742 GluR5 758 GluRD 742 GluRC 745 GluRB 741 GluRA GluR5 ELKQ Fringe shift Rdius (cm) Tyr490 J * * * Figure 2 Protein engineering in the dimer interfce does not generte nondesensitizing kinte receptors. () Crystl structure of the wild-type GluR5 dimer interfce, showing the interction etween helices D nd J. Electron density for s A -weighted F o F c omit mps t 1.9-Å resolution, contoured t 3.7 s ws clculted with the indicted side chins nd wter molecule omitted from the F c clcultion. Dshed lines indicte hydrogen onds. Tyr506 projects out of the dimer interfce. () Amino cid sequence lignment of helices D nd J in AMPA nd kinte receptors; conserved differences in the residues tht contriute to dimer formtion in the GluR2 L483Y mutnt re shown in color. (c) Rdil sornce profiles of wild-type (WT) GluR5 R5 in sedimenttion equilirium t rotor speeds of 15,000 r.p.m. (lck squres), 22,000 r.p.m. (red circles) nd 30,000 r.p.m. (green tringles). Solid line is the fit with model for monomer-dimer self-ssocition where dimer is undetectle; sttisticl nlysis indictes the K d is 46 mm; the highest oserved protein concentrtion ws B120 mm. Lower chrt shows residuls of the fit. (d) Sedimenttion equilirium for the GluR5 ELKQ mutnt, colored s for wild-type GluR5; for clrity, only every tenth dt point is shown. Highest oserved concentrtion ws B124 mm. Sttisticl nlysis for fit of monomer-dimer equilirium indictes K d 4 8 mm. Lower chrt shows the residuls of the fit. (e) Responses recorded from outside-out ptches from HEK cells trnsfected with the indicted cdna species; the onset of desensitiztion is fit with single-exponentil functions (red) nd is only two-fold slower for GluR6 ELKQ thn for wild-type GluR6. (f) Crystl structure of the GluR6 ELKQ mutnt dimer interfce, showing the interction etween helices D nd J. Electron density for s A -weighted F o F c omit mps t 1.9-Å resolution, contoured t 3.2 s ws clculted with the indicted side chins omitted from the F c clcultion. Tyr490 projects into the dimer interfce. muttions. Western lotting nlysis reveled tht dimer formtion requires cysteine residues in oth helices D nd J, s single cysteine mutnts formed receptors of similr moleculr weight to wild-type GluR6 (Fig. 3). Cossemly of the GluR5 nd GluR6 cross-linked lignd-inding cores s dimers of dimers ws mesured y nlyticl ultrcentrifugtion. These experiments reveled tht for oth GluR5 (Fig. 3c) nd GluR6 (Fig. 3d), the K d for the dimer-tetrmer equilirium is t lest 5 mm. Thus, the lignd-inding core dimers of kinte receptors hve miniml tendency to form tetrmeric ssemly in the sence of the N-terminl domin nd the ion chnnel. However, we note tht wek interctions etween dimer pirs could e functionlly significnt in the context of n intct receptor emedded in the twodimensionl environment of the lipid ilyer. Another importnt cvet is the possiility tht the disulfide cross-links introduced for these experiments pertur the formtion of dimers of dimers, ecuse, s descried elow, crystllogrphic nlysis showed tht the orienttion of the protomers in cross-linked dimer ws slightly different from tht in wild-type receptors. Non-desensitizing AMPA nd kinte receptors Hving estlished tht disulfide ond cross-links etween helices D nd J re formed in kinte receptors, we exmined the functionl consequences using electrophysiologicl techniques. We lso constructed the corresponding cross-linked form of the AMPA receptor GluR2 suunit. Control wild-type receptor responses to 10 mm L-glutmte showed rpid desensitiztion, which ws essentilly complete t equilirium nd similr for AMPA nd kinte receptors (Fig. 4, left column). The time constnts nd percent desensitiztion t 500 ms were s follows: GluR2, 6.9 ± 0.9 ms, 98.3 ± 0.5% (n ¼ 7); GluR5, 1.0 ± 0.09 ms, 99.7 ± 0.3% (n ¼ 4); GluR6, 4.3 ± 0.1 ms, 99.1 ± 0.2% (n ¼ 12); GluR7, 5.2 ± 0.51 ms, 99.5 ± 0.2% (n ¼ 4). In contrst, for ll three kinte receptors, s well s for GluR2, the disulfide ond cross-linked constructs were lmost completely 1122 VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

4 c Fringe units M w ph 5 GluR5 Y506C L768C ph 8 Ox. DTT Ox Rdius (cm) DTT non-desensitizing (Fig. 4, right column). For responses to 10 mm L-glutmte mesured t 500 ms, the extents of desensitiztion were s follows: GluR2, 5.5 ± 2.1% (n ¼ 4); GluR5, 7.4 ± 1.9% (n ¼ 4); GluR6, 7.5 ± 0.8% (n ¼ 12); GluR7, 18.3 ± 1.7% (n ¼ 4). For comprison, we lso mesured responses for the GluR2 L483Y mutnt, which showed 6.6 ± 2.0% (n ¼ 4) desensitiztion t 500 ms, similr to tht for cross-linked receptors. Although our experiments were designed to lock desensitiztion y introduction of conformtionl restrints, it is possile tht the loss of desensitiztion could hve resulted insted from lrge reduction in gonist ffinity. Such mechnism ws excluded y dose-response nlysis for the mutnt, which gve concentrtion for the hlf-mximum response (EC 50 ) of 33.8 ± 2.0 mm glutmte (n ¼ 12), 20-fold lower thn the pulished vlues of 762 nd 694 mm for wild-type GluR6 (refs. 25,26). This indictes tht the loss of desensitiztion in the mutnt does not result from lower ffinity for glutmte. The high ffinity of the mutnt ws not unexpected, s for AMPA receptors the non-desensitizing GluR2 L483Y mutnt produces 70-fold decrese in EC 50 compred with wild-type GluR2 (refs. 27,28). d Fringe units RNA WT Rdius (cm) Y490C L752C Ox. Y490C L752C DTT Y490C L752C M w Figure 3 Lignd-inding domin dimer formtion resulting from disulfide ond cross-links. () SDS PAGE for the purified GluR6 lignd-inding core Y490C L752C mutnt, stored overnight t either ph 5 or ph 8; smples were run in gel-loding uffer either without (Ox.) or with 100 mm DTT; for crystlliztion nd nlyticl ultrcentrifugtion, the dimer species ws isolted y gel-permetion chromtogrphy. Left lne contins moleculr weight mrkers. () Western lot of memrne frctions from uninjected Xenopus oocytes ( RNA) nd oocytes expressing either wild-type (WT) GluR6, without DTT (Y490C L752C Ox.); GluR6 Y490C L752C with 100 mm DTT (Y490C L752C DTT), GluR6 Y490C (Y490C) or GluR6 L752C (L752C). Right lne contins moleculr weight mrkers. (c,d) Sedimenttion equilirium nlysis for the GluR5 Y506C L768C cross-linked lignd-inding core dimer (c) nd the GluR6 Y490C L752C cross-linked lignd-inding core dimer (d) using rdil fringe displcement, colored s in Figure 2. The highest oserved dimer concentrtions were B48 mm for GluR5 nd B52 mm for GluR6; solid lines re est-fit distriutions for dimer-tetrmer model; sttisticl nlysis indictes K d 4 10 mm for GluR5 nd K d 4 15 mm for GluR6. less thn the vlue for wild-type GluR6. The ltter effect is proly due to residul frction of receptors with intct disulfide onds in the dimer interfce, nd not to clevge of Cys-Cys onds elsewhere in the receptor, s ppliction of DTT to wild-type receptors hd little effect on desensitiztion (dt not shown). Shorter pplictions of DTT produced responses with rpid ut incomplete desensitiztion, which proly results from formtion of mixed popultion of receptors with reduced nd intct cross-links (Fig. 5). Supporting this, we found tht in few exceptionlly stle ptches, it ws possile to reversily introduce nd remove desensitiztion y sequentil pplictions of DTT nd CuPhen (Fig. 5). We WT GluR2 10 mm L-Glu WT GluR5 10 mm L-Glu GluR2 L483C L751C 20 pa 10 pa GluR5 Y506C L768C Restortion of desensitiztion y DTT To verify tht the locking of desensitiztion resulted from restrined conformtionl moility, we exmined the effect of reducing gents, with the ide tht receptors should desensitize with norml kinetics once the disulfide ond is roken. Appliction of 30 mm DTT to cells for 10 min efore recording ws required to consistently restore desensitiztion in the GluR6 cross-linked mutnt (Fig. 5). The time constnt of desensitiztion fter tretment with DTT, 1.3 ± 0.1 ms (n ¼ 5), ws three-fold fster thn for wild-type GluR6, wheres t 500 ms the extent of desensitiztion reched 92.6 ± 5.1%, only slightly 100 pa WT GluR6 10 mm L-Glu 20 pa 20 pa 10 pa WT GluR7 10 mm L-Glu GluR7 H492C L753C Figure 4 Non-desensitizing glutmte receptors creted y lignd-inding core disulfide ond cross-links. Responses to 10 mm glutmte, recorded from outside-out ptches from HEK cells trnsfected with the indicted cdna species, re shown for wild-type GluR2 nd the kinte receptors GluR5, GluR6 nd GluR7 (left chrts) nd for their lignd-inding core doule cysteine mutnts (right chrts). Upper trces in ech pnel show open-tip responses recorded t the end of the experiment. 100 ms 50 pa 15 pa NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER

5 10 mm L-glutmte 10 pa 30 mm DTT 10 min 100 ms 30 mm DTT 5 min 10 µm CuPHEN 5 min 30 mm DTT 5 min 30 pa 50 pa 50 pa 50 pa suggest tht the resistnce of to DTT results from comintion of effects: limited ccess to the dimer interfce in the intct receptor, nd the need to rek two sets of disulfide onds ecuse of the two-fold moleculr symmetry of the dimer ssemly. After reduction y DTT, the functionl properties of receptors hroring cysteine pirs on helices D nd J were similr ut not identicl to those of wild-type GluR6, most proly owing to differences in size nd chemistry of the ntive tyrosine nd leucine side chins nd the mutted cysteine side chin. Receptors with single cysteine muttions lso showed chnges in the kinetics of onset of desensitiztion. For the GluR6 Y490C mutnt, the time constnt of desensitiztion ws 45.5 ± 4.8 ms, ten-fold slower thn for wild-type GluR6, lthough t 500 ms desensitiztion ws nerly complete, 99.6 ± 0.2% (n ¼ 11). These reltively sutle effects most proly result from minor perturtions in the dimer interfce, s prior work on oth AMPA nd kinte receptors shows tht this cn result in either smll increses or smll reductions in the rte of onset of desensitiztion 7,15. For GluR6 L754C, we were unle to otin functionl responses in outside-out memrne ptches. c % desensitiztion R2 R5 R6 Wild-type Cys cross-link R7 R2 R5 R6 R7 R6 + DTT R2 L483Y Figure 5 Modultion of desensitiztion y disulfide ond clevge. () Responses to 10 mm L-glutmte recorded from outside-out ptches from cells expressing the mutnt efore (left) nd fter (right) tretment with 30 mm DTT for 10 min. () Sequentil responses to 10 mm L-glutmte, recorded in single ptch fter tretment with 30 mm DTT for 5 min (left) followed y 10 mm CuPhen for 5 min (middle) nd 30 mm DTT for 5 min. (c) Summry of the percent desensitiztion recorded from outside-out ptches 500 ms fter the strt of the ppliction of 10 mm L-glutmte, for the indicted constructs, including GluR2 L483Y; error rs show s.e.m. (P 4 0.5) from tht of the GluR2 L483Y mutnt (Fig. 5c). To further test this hypothesis, we solved the structure of the GluR6 Y490C L752C cross-linked dimer. There were 100 ms two molecules in the symmetric unit, linked y pir of disulfide onds nd relted y two-fold xis of symmetry (Fig. 6); electron density for the disulfide onds ws clerly visile in omit mps (Fig. 6). The resulting dimer ws similr to those of GluR2 L483Y nd the GluR6 ELKQ mutnt, ut with some importnt differences. The uried surfce re of 686 Å 2 per suunit ws less thn for GluR6 ELKQ (1,009 Å 2 per suunit). A lest-squres superposition of the dimer structure on the GluR6 ELKQ nd GluR2 L483Y mutnt dimers gve r.m.s. devitions of 1.75 nd 1.51 Å, respectively, when C toms from oth suunits were fit simultneously; these decresed to only 0.31 nd 0.58 Å, respectively, when protomer A ws fit lone, nd 0.25 nd 0.58 Å, respectively, when protomer B ws fit lone. These differences occur ecuse ech of the suunits in the cross-linked dimer hs rotted 61 out n xis running close to the center of mss of ech suunit, pproximtely perpendiculr to the moleculr two-fold xis of symmetry. The seprtion t the top of domin 1, ner the N terminus of helix J (mesured s the distnce etween the C toms of Ser741 in ech suunit) therefore increses from 16.2 Å in the GluR6 ELKQ mutnt dimer structure (Fig. 6c)to20.2Åin the Structurl nlysis of the dimer The nerly complete lock of desensitiztion y formtion of disulfide ond cross-links etween helices D nd J suggests tht the ligndinding core dimer gonist complexes re trpped in their ctive sttes. Consistent with this, the extents of desensitiztion for crosslinked GluR2, GluR5 nd GluR6 were not significntly different 20.2 Å Figure 6 Anlysis of disulfide ond cross-linked receptors. () Crystl structure of the mutnt cross-linked dimer ssemly. C positions of Ser761 nd Ile653 re show s lck spheres t the top nd ottom of ech suunit, respectively. () Electron density for s A -weighted F o F c omit mps t 2.25-Å resolution, contoured t 3.25 s clculted with the Y490C L752C residues omitted from the F c clcultion. (c) Crystl structure of the GluR6 ELKQ mutnt dimer, illustrting the chnge in seprtion of Ser761 nd Ile653 compred with the disulfide ond crosslinked receptor. (d) Superimposed responses to 10 mm glutmte (lck rs ove trces) recorded in the sence or presence of cyclothizide (white r; sterisks mrk trces in the presence of cyclothizide), from outside-out ptches from HEK cells trnsfected with cdnas for GluR2 L483C L752C (left) nd GluR2 L483Y (right). c 33.4 Å GluR6 ELKQ 16.2 Å 38.0 Å d GluR2 L483C L751C L483Y * * 30 pa 250 ms 1124 VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

6 cross-linked dimer (Fig. 6). By contrst, t the se of the dimer, pproching the plne of the memrne, the seprtion etween the linkers leding to the trnsmemrne segments (mesured s the distnce etween the C toms of Ile653 in domin 2) decreses from 38.0 Å in the GluR6 ELKQ mutnt dimer structure to 33.4 Å in the cross-linked dimer. For oth AMPA nd kinte receptors, there is now sustntil evidence tht prtil gonists induce intermedite degrees of closure of the lignd-inding cores, resulting in grded extension of the seprtion of the linkers tht led to the trnsmemrne segments in dimer ssemly 21, Hence, we would expect glutmte to e less efficcious gonist in these constructs, owing to the decrese in seprtion of the trnsmemrne linkers resulting from the pivoting of ech suunit reltive to the dimer interfce. The effect of this pivoting, if domin 1 is constrined to the ctive conformtion found in the GluR6 ELKQ mutnt structure (Fig. 6c), is equivlent to 61 opening of the lignd-inding core in ech suunit. This is out twice the opening produced y the prtil gonist kinic cid 31, which produces 50% of the response to glutmte 15. To test the ide tht full-length disulfide ond cross-linked receptors re relxed compred with the ctive dimer conformtion, we exmined the ction of cyclothizide, n llosteric modultor of AMPA receptors, on the GluR2 L483C L751C mutnt. Prior crystllogrphic studies revel tht cyclothizide inds to pir of two-fold relted sites locted t the se of the GluR2 lignd-inding domin dimer nd cts s moleculr wedge tht stilizes the dimer in its ctive conformtion 6. As control experiment, we tested the effect of cyclothizide on the GluR2 L483Y mutnt, which should not e modulted y cyclothizide, s it is lredy locked into the fully ctive conformtion 6. The results of this experiment were noteworthy (Fig. 6d): we oserved 2.8 ± 0.25 fold (n ¼ 3) potentition of responses to 10 mm L-glutmte y 100 mm cyclothizide for GluR2 L483C L751C, wheres for GluR2 L483Y, responses to glutmte showed wek inhiition, with cyclothizide/control response rtio of 0.9 ± 0.06 (n ¼ 4). The potentition of the response y cyclothizide ws not due to chnge in gonist potency, s responses to 30 mm glutmte showed neither n increse in response mplitude compred with the response t 10 mm glutmte nor reltive chnge in potentition y cyclothizide (dt not shown). DISCUSSION The lock of AMPA- nd kinte receptor desensitiztion y intermoleculr disulfide onds etween the domin 1 surfces of their lignd-inding cores provides further evidence tht the dimer crystl structures solved for glutmte receptor gonist complexes for GluR2, GluR5 nd GluR6 correspond to the ctive, non-desensitized conformtion. This result lso estlishes tht desensitiztion is withindimer phenomenon, ecuse, lthough the protomers within dimer pir were locked together, no constrints were introduced to prevent movements of the dimer pirs reltive to ech other. This is consistent with the pprent two-fold symmetry oserved in some, ut not ll, single-prticle nlyses of intct AMPA receptor ion chnnels 32,33.The GluR2 L483Y mutnt nd the disulfide ond cross-linked GluR2, GluR5, GluR6 nd GluR7 hd similr effects in locking desensitiztion of AMPA nd kinte receptors; this indictes tht, despite differences in their sensitivity to llosteric modultors nd the unpredictle effect of muttions within the dimer interfce, the two receptor sutypes shre conserved moleculr mechnisms of ctivtion nd desensitiztion. In contrst, different mechnisms seem to operte in NMDA receptors. Notly, when disulfide onds were engineered into equivlent positions in the NMDA receptor NR1 nd NR2A suunits to crete disulfide-linked heterodimers, reducing gents did not introduce desensitiztion, s found for the AMPA nd kinte receptor mutnts, ut insted potentited gonist responses y n unknown mechnism 22. The GluR5 nd GluR6 ELKQ muttions did not introduce mesurle dimer formtion of the solule lignd-inding cores, in contrst to the high-ffinity dimer formtion tht occurs with the GluR2 L483Y mutnt 6. Introduction of tyrosine residues t the equivlent positions in the NR1 suunit ut not the NR2A suunit lso promotes homodimer formtion. Crucilly, the fce of helix J, which forms the inding site for the tyrosine side chin in the GluR2 L483Y nd GluR6 ELKQ mutnt crystl structures, contins the required leucine-lysine sndwich in NR1, wheres in NR2A the lysine hs een replced y the glutmine found in kinte receptors. The finding tht the GluR5 nd GluR6 ELKQ muttions do not chnge the K d for dimer formtion is noteworthy, ecuse this excludes the possiility tht the strong desensitiztion oserved for the GluR6 ELKQ mutnt is consequence of the much higher stility of the desensitized stte, indicted y the 125-fold slower rte of recovery from desensitiztion for GluR6 compred with GluR2 (ref. 34). We see lmost no desensitiztion for the disulfide ond crosslinked dimers of GluR2, GluR5, GluR6 nd GluR7, even though in the crystl structure of the mutnt, the dimer hs relxed compred with wht we elieve to e the fully ctivted stte. One interprettion of this result is tht desensitiztion is n ll-ornone process requiring lrge movements tht not re possile in the disulfide ond constrined dimers. An eqully likely explntion is tht the cross-linked receptors re lredy in prtilly relxed stte nd tht no further movements occur in the dimer interfce fter ctivtion y glutmte. Low-resolution single-prticle nlysis revels tht AMPA receptors cn smple multiple conformtions such tht the extrcellulr domins move over lrge distnces 35 ; it is plusile tht some of these correspond to the ctive nd desensitized sttes. This is reinforced y recent crystllogrphic studies of AMPA receptor lignd-inding cores, which suggest tht desensitiztion results from 201 rottion of the suunits in dimer pir, resulting in 9-Å increse in seprtion t the top of the dimer ssemly nd n 11-Å decrese in seprtion t the se of the dimer 36. These chnges re nerly 2.5-fold lrger thn those oserved for the GluR6 Y490C L752C mutnt dimer. Our experiments shed further light onto the interply of receptor ctivtion nd desensitiztion. In the consensus model for AMPA receptor ctivtion, extension of the linkers leding to the trnsmemrne segments controls the degree of receptor ctivtion, nd there is n pproximtely liner reltionship etween linker movement nd gonist efficcy 27,30,37. Consistent with this model, nd with the relxed conformtion of the mutnt dimer crystl structure, we found tht the llosteric modultor cyclothizide potentited responses to glutmte for the disulfide cross-linked GluR2 mutnt ut not for GluR2 L483Y. We elieve this occurs ecuse cyclothizide locks the dimer into its fully ctive conformtion. Allosteric lignds for kinte receptors hve not een reported, ut we predict tht if these ecome ville, we will see potentition of responses for s well. In summry, we hve creted series of non-desensitizing kinte receptors y introducing conformtionl restrints vi intermoleculr disulfide cross-links. These were designed on the sis of crystl structures for wild-type GluR2, GluR5 nd GluR6. The success of this pproch suggests tht AMPA nd kinte receptors hve conserved gting mechnisms nd tht, to desensitize, their ligndinding cores must undergo lrge conformtionl rerrngements. NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER

7 METHODS Mutgenesis nd iochemistry. Point muttions for electrophysiologicl experiments were introduced y overlp PCR with Pfu DNA polymerse to generte complementry DNA frgments; these were ligted into ppropritely digested GluR2, GluR5, GluR6 nd GluR7 prk vectors engineered to produce green fluorescent protein vi downstrem internl riosome entry site, s descried previously 6. After sucloning, the mplified segments were sequenced in their entirety. The sme constructs were inserted into pgem/he for preprtion of mrna trnscripts using T7 RNA polymerse (Amion mmessge mmchine). Muttions in the lignd-inding cores of GluR6 were produced using the sme pproch, ut with modified pet vectors for protein expression in E. coli followed y nickel-nitrilotricetic cid nd ion-exchnge chromtogrphy s descried 31. For the disulfide ond cross-linked GluR5 nd GluR6 constructs, we used n dditionl step of dilysis t ph. 8.0 for 1 2 d, followed y preprtive gel-permetion chromtogrphy using Superose 12 XK 26/70 column to remove residul monomeric protein nd higher moleculr weight ggregtes. ESI TOF mss spectroscopy ws used to confirm the identities of purified proteins. Amino cid numers correspond to the mture protein fter clevge of the signl peptide. Western lotting nlysis ws performed using the memrne frction from oocytes, soluilized in uffer contining 20 mm Tris-HCl (ph 8.0), 50 mm NCl, 1% (w/v) -dodecylmltoside, 0.3% (w/v) CHAPS, complete protese inhiitor cocktil (Roche) nd 2 mm N-ethylmleimide. The primry ntiody ws monoclonl nti-glur6 (residues ; Chemicon ), used t 1:5,000 dilution; the secondry ntiody ws HRP-linked nti-rit IgG (Amershm), used t 1:2,000; lots were developed with ECL (Amershm) nd imged using cooled CCD cmer. Anlyticl ultrcentrifugtion. Sedimenttion equilirium experiments were conducted with n Optim XLI/A centrifuge (Beckmn Coulter). Smples (180 ml) were loded t concentrtions of 0.1, 0.3 nd 1 mg ml 1 for wild-type GluR5 nd GluR6 nd their ELKQ mutnts, nd t 1 mg ml 1 for the GluR5 nd GluR6 cross-linked dimers, in uffer contining 150 mm NCl, 20 mm sodium phosphte (ph 7.2), 1 mm EDTA nd 2 mm glutmte. Sedimenttion equilirium ws ttined t rotor temperture of 10 1C t sequentil rotor speeds of 15,000, 22,000, nd 30,000 r.p.m., which estlished concentrtion grdients rnging from 0 to B2 mgml 1, corresponding to concentrtions of B125 mm for wild-type receptors nd ELKQ mutnts, nd 50 mm for the crosslinked dimers. Asornce opticl detection t 280, 250 nd 230 nm ws used. To increse precision of the nlysis for the cross-linked dimers nd ELKQ vrints, Ryleigh interference optics were lso used. For interference detection, spphire windows nd ged cell components were used. The dt were nlyzed with SEDPHAT 38 y glolly fitting dt sets for ech construct, cquired with different rotor speeds nd detection systems, using soft mss conservtion nd explicit lgeric time-invrint noise decomposition 38.The monomer uoynt molr mss vlues Mð1 vrþ were included s floting prmeters nd converged close to the vlues of B7,600 D expected from the mino cid sequence, within the uncertinty of the prtil-specific volume. Error limits for the ssocition constnts were determined y exploring projections of the error surfce with F-sttistics. The homogeneity of the smples ws exmined in prllel experiments y sedimenttion velocity t protein concentrtions of 1.5 mg ml 1 using the c(s) method 39, resulting in uniform peks comprising 499% nd 498.3% of mteril for wild-type GluR5 nd GluR6, respectively; 499% nd 98.1% for their ELKQ mutnts; nd 499% nd 98.2% for the GluR5 nd GluR6 dimers. This further estlished the sence of detectle self-ssocition of the ELKQ mutnts, s indicted y the Svederg vlue of 2.689S for GluR5 ELQK, which ws virtully identicl to tht for wild-type GluR5 (2.687S). Structure determintion. Crystls were grown t 277 K using the hnging drop technique nd reservoir of 1.5 M mmonium sulfte, 0.1 M NCl, M sodium cette nd 0.1 M HEPES (ph 7.2) for the GluR6 ELKQ mutnt, or 20% (w/v) PEG 4,000, 20% (v/v) isopropnol nd 0.1 M sodium citrte (ph 5.6) for the dimer. The protein solutions contined 20 mm NCl, 10 mm sodium glutmte, 1 mm EDTA nd 10 mm HEPES (ph 7.0). GluR6 ELKQ crystls were cryo-protected y rief soks in mother liquor supplemented with incresing concentrtions of glycerol to finl vlue of 18% (v/v). X-ry diffrction dt were collected t Advnced Photon Source Tle 1 Dt collection nd refinement sttistics GluR6 ELKQ Dt collection Spce group P P4 Cell dimensions,, c (Å) 94.5, 94.5, , 108.8, 47.4 Resolution (Å) ( ) ( ) R merge (0.452) (0.437) I / si 13.0 (3.8) 10.1 (2.9) Completeness (%) 99.9 (100) 98.9 (100) Redundncy 6.5 (6.4) 3.1 (3.0) Refinement Resolution (Å) No. reflections 72,632 26,406 R work / R free 17.2 / / 24.4 No. toms Protein 6,287 4,045 Lignd Wter B-fctors Protein Lignd Wter R.m.s. devitions Bond lengths (Å) Bond ngles (1) Vlues in prentheses re for highest-resolution shell. emline I2 t 100 K using MAR 300 CCD detector (Tle 1). Dt ws indexed, scled nd merged using HKL2000 (ref. 40). Both structures were solved y moleculr replcement with MOLREP 41 using wild-type GluR6 monomer (PDB 1S7Y) s serch proe nd refined with REFMAC 5 (ref. 41) using three TLS groups per suunit identified using motion-determintion nlysis 42. Model uilding into s A -weighted F o F c nd 2F o F c nd composite omit mps ws done using O 43 nd coupled with cycles of crystllogrphic refinement, resulting in R work vlues of 17.2% nd 17.2%, nd R free vlues of 21.1% nd 24.4%, for the GluR6 ELKQ mutnt nd GluR6 Y490C L752C dimer, respectively (Tle 1). Additionl crystllogrphic clcultions were performed using CCP4 (ref. 41) nd the USF suite 44. Domin closure ws clculted fter lest-squres superposition of domin 1 using C toms s descried 31. Dimer-contct surfce res were clculted using AREAIMOL 41 with point density vlue of 20. Figures were prepred using MOLSCRIPT 45, RASTER3D 46 nd PyMOL ( Electrophysiologicl experiments. HEK-T cells were trnsfected with 1.5 mg of cdna per 35-mm coverslip y the clcium-phosphte method 47 ; experiments were performed t room temperture h fter trnsfection. Cells were identified y green fluorescence nd outside-out memrne ptch recordings performed with n Axoptch 200A mplifier. Ptches were positioned in front of fst perfusion system consisting of glss flow pipe mounted on piezoelectric trnsltor 48,49. At the completion of recording, solutionexchnge time (20% 80% to pek) ws estimted from open-tip responses nd rnged from ms. Ptch pipettes hd resistnce of 2 4 MO nd were filled with solution contining 150 mm CsF, 20 mm HEPES, 10 mm NCl nd 10 mm EGTA (ph 7.3). The extrcellulr medium contined 140 mm NCl, 2.4 mm KCl, 10 mm HEPES, 10 mm glucose, 4 mm MgCl 2, 4 mm CCl 2 (ph 7.3, 305 mosm). Cyclothizide ws dissolved in DMSO efore dilution with extrcellulr solution to finl concentrtion of 100 mm. Holding potentil ws typiclly 70 mv. Currents were filtered t 5 khz nd digitized t 10 khz. The dt were nlyzed using Axogrph 4.9 (Moleculr Devices). For mesurements of dectivtion nd desensitiztion, the decy time course of the response fter 1-ms ppliction or during 500-ms ppliction 1126 VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

8 of 10 mm glutmte ws fit to single-exponentil function. To mesure recovery from desensitiztion, pired-pulse protocol ws used nd the pek mplitude of the second pulse ws clculted s rtio of the first pulse. Accession codes. Protein Dt Bnk: Coordintes nd structure fctors for the GluR6 ELKQ mutnt nd dimer hve een deposited with ccession codes 2I0B nd 2I0C, respectively. ACKNOWLEDGMENTS We thnk C. Glsser for technicl ssistnce, P. Seeurg (Mx Plnck Institute for Medicl Reserch, Heidelerg) nd S. Heinemnn (Slk Institute for Biologicl Studies) for the gift of the wild-type GluR plsmids, H. Deng for help with cell culture, M. Zhng for help with protein purifiction nd crystlliztion, H. Jffe (Protein/Peptide Sequencing Fcility, Ntionl Institute of Neurologicl Disorders nd Stroke) for mss spectrl nlysis, E. Gouux for shring results efore puliction nd A. Plested for reding the mnuscript. Nucleic cid sequencing ws performed y the Ntionl Institute of Neurologicl Disorders nd Stroke DNA sequencing fcility, Porter Neuroscience Reserch Center. This work ws supported y the Brown foundtion (C.R.), Neuroscience Trining grnt T32 GM (M.C.W.) nd the intrmurl reserch progrm of the Ntionl Institute of Child Helth nd Humn Development, US Ntionl Institutes of Helth, Deprtment of Helth nd Humn Services (M.L.M.). AUTHOR CONTRIBUTIONS Electrophysiologicl experiments were performed y M.C.W., nlyticl ultrcentrifugtion y P.S., iochemistry nd crystllogrphy y M.L.M., moleculr iology nd iochemistry y A.G. Dt ws interpreted nd the pper written y M.L.M., C.R. nd M.C.W. COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Hille, B. Ionic chnnels of excitle memrnes 3rd edn. (Sinuer Assocites, Sunderlnd, Msschusetts, USA, 2001). 2. Jones, M.V. & Westrook, G.L. 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J. Physiol. (Lond.) 458, (1992). NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER