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Home , AMPA, AMPA receptor, Kainate receptor, Willardiine

Subunit-selective iGluR antagonists can potentiate heteromeric responses by blocking desensitization

Stefan Polloka and Andreas Reinera,1

aDepartment of Biology and Biotechnology, Ruhr University Bochum, 44801 Bochum, Germany

Edited by Ehud Y. Isacoff, University of California, Berkeley, CA, and approved August 31, 2020 (received for review April 18, 2020) Ionotropic glutamate receptors (iGluRs) are key molecules for treating (LY5454694) (17). In the case of AMPA receptors, synaptic signaling in the , which makes which have more closely related -binding domains (LBDs), them promising drug targets. Intensive efforts are being devoted some moderately selective have been reported, but to the development of subunit-selective , which should en- subunit-selective antagonists are rare (18). able more precise pharmacologic interventions while limiting the Subunit-specific ligands are thought to allow for more precise effects on overall neuronal circuit function. However, many AMPA intervention, as their action remains confined to a small set of and complexes are heteromers composed receptor subtypes and to the regions where these subtypes of different subunits. Despite their importance, little is known are expressed. However, many or even most neuronal iGluRs are about how subunit-selective ligands affect the gating of hetero- heteromeric receptors (3, 4, 19–23), which increases the versa- meric iGluRs, namely their activation and desensitization proper- – ties. Using fast ligand application experiments, we studied the tility of iGluR signaling (24 26) and allows for fine-tuned reg- effects of competitive antagonists that block glutamate from bind- ulation. This motivated us to investigate how heteromeric kainate ing at part of the four subunits. We found that UBP-310, a kainate and AMPA receptors are impacted by subunit-specific ligands. with high selectivity for GluK1 subunits, re- One hallmark of AMPA and kainate receptor gating is their duces the desensitization of GluK1/GluK2 heteromers and fully fast (millisecond) desensitization, which is induced by prolonged

abolishes the desensitization of GluK1/GluK5 heteromers. This ef- glutamate binding. Desensitization is tightly regulated by, for ex- PHARMACOLOGY fect is mirrored by subunit-selective agonists and heteromeric re- ample, RNA splicing (27) and auxiliary subunits (28–31). Depend- ceptors that contain binding-impaired subunits, as we show for ing on the glutamate dynamics in the synaptic cleft, desensitization both kainate and GluA2 AMPA receptors. These findings are con- exerts important physiological functions. It can terminate the cur- sistent with a model in which incomplete occupancy at the rent flow after glutamate release and can shape the responses to low four receptor subunits can provide activation without inducing residual concentrations of glutamate (e.g., refs. 32, 33). Recovery desensitization. However, we did not detect significant steady-state from desensitization is slow, taking tens of milliseconds in the case currents during UBP-310 dissociation from GluK1 homotetramers, in- of AMPA receptors but seconds for kainate receptors (34). De- dicating that antagonist dissociation proceeds in a nonuniform and sensitization and recovery may thus control the responses to sub- cooperativity-driven manner, which disfavors nondesensitizing occu- pancy states. Besides providing mechanistic insights, these results sequent release events and may affect postsynaptic short-term have direct implications for the use of subunit-selective antagonists plasticity and frequency dependence (29). Suppression of AMPA in neuroscience research and envisioned therapeutic interventions. receptor desensitization causes severe developmental defects (35).

ligand-gated channel | medicinal chemistry | non-NMDA receptor | Significance receptor modulation Ionotropic glutamate receptors (iGluRs) are a diverse family of lutamate is an important in the mamma- tetrameric ligand-gated ion channels. Recent structural infor- Glian brain. Its excitatory action is mediated by ionotropic mation has provided valuable insight into the conformational glutamate receptors (iGluRs), a family of tetrameric ligand-gated dynamics and assembly properties of iGluRs, most importantly ion channels comprising 18 different (1, 2). The different on heteromeric iGluRs, which are prevalent in the nervous iGluR subtypes play key roles in synaptic transmission, modula- system. However, little is known about how channel gating is tion, and plasticity (3–5); thus, controlling iGluR function may be controlled by the 4 subunits within the tetramers, different li- useful for treating a wide range of diseases, such as pain, migraine, gand occupancies, and intersubunit cooperativity. In this study, , mood disorders, and , as well as for managing we probe how subunit-selective ligands affect activation and desensitization of heteromeric iGluRs and we provide mecha- ischemic stroke, glioblastoma, and neurodegeneration. nistic insight into ligand dissociation. Our findings have direct The discovery of agonists and antagonists that discriminate pharmacologic implications, as we show that subunit-selective between the AMPA (GluA), kainate (GluK), and NMDA (GluN) antagonists can block desensitization of heteromeric iGluRs, receptor subfamilies (1, 6) was a key step in dissecting their thereby causing potentiation instead of inhibition. physiological functions. However, the clinical utility of iGluR ag- onists and antagonists has remained limited, because broad acti- Author contributions: S.P. and A.R. designed research; S.P. performed research; S.P. and vation or inhibition of iGluRs is associated with severe side effects A.R. analyzed data; and S.P. and A.R. wrote the paper. (e.g., refs. 7–9). To overcome these side effects, current medicinal The authors declare no competing interest. research is focusing on allosteric ligands with modulatory effects This article is a PNAS Direct Submission. and subunit-specific ligands that show selective binding at only one Published under the PNAS license. or a few iGluR subtypes (10–12). For instance, subunit-selective 1To whom correspondence may be addressed. Email: [email protected]. kainate receptor antagonists have been proposed for treating ce- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ rebral (LY377770) (13), (LY382884) (14), and doi:10.1073/pnas.2007471117/-/DCSupplemental. epilepsy (UBP-310) (15, 16) and have been in clinical trials for

www.pnas.org/cgi/doi/10.1073/pnas.2007471117 PNAS Latest Articles | 1of8 Downloaded by guest on September 29, 2021 Recent full-length iGluR structures provide insight into the which became part of some widely used models to describe iGluR conformational changes occurring on ligand binding (36–39), but gating (26, 47). However, the interpretation of experiments with how the ligand occupancy at the four LBDs controls receptor low ligand concentrations is complicated by slow binding kinetics activation and desensitization remains unclear. Structure-function and the fact that mid- and high-occupancy states are still sampled studies have revealed that glutamate binding leads to closure of occasionally (48). the clamshell-like LBDs, which appears to be a requirement for More recent work on a special class of heteromeric kainate efficient activation (40). Single-channel recordings on desensitization- receptors—those incorporating both low- and high-affinity sub- blocked receptors have demonstrated that at least two subunits must units (e.g., GluK2 and GluK5, respectively; formerly known as be occupied by agonists for substantial activation to occur (31, 41). GluR6 and KA2)—has shown that these receptors can evade Desensitization is thought to originate from rearrangements of the desensitization with two occupied subunits while showing sub- interface between two adjacent LBDs, as stabilization of this interface stantial activation (48–50). In these experiments, subunit-selective can slow or abolish desensitization (42–45). agonists (48, 51), the incorporation of mutated subunits with Based on the observation that even low glutamate concen- lowered affinity (49, 50), and tethered photoswitchable ligands trations elicit efficient desensitization in the absence of significant (48) were used to ensure less than full receptor occupancy. activation (31, 46, 47), it was further concluded that a single oc- However, whether this behavior also applies to other types of cupied subunit may be sufficient to cause receptor desensitization, kainate receptors and the postsynaptically far more abundant

A

4 3:1 2:2 1:3 4

GluK1 GluK1/GluK2 heteromers GluK2

B GluK1 GluK1/GluK2 GluK2 1 μM UBP-310 10 μM UBP-310 10 μM UBP-310 3 mM Glu 3 mM Glu 3 mM Glu

Iss / Ipeak= 1.2 % Iss / Ipeak= 26 %

τdes= 0.7 ms τdes= 2.1 ms τdes= 3.9 ms τdes= 3.2 ms τdes= 3.0 ms

A UPB / A Glu= 0.23 25 pA 50 pA 25 pA 5 ms 20 ms 5 ms

C K1 K1/2 K2 DFK1 K1/2 K2 E K1 K1/2 K2 K1/2 10 μM UBP-310 (7) (17) (8) (7) (17) (8) (7) (17) (8) (37)(17) 1.2 *** *** ns *** ns ** ns 80 1.0 30 6 60 0.8 (%) (%) 20 4 (rel.)

0.6 (ms) 40 peak peak I I

/ des /

peak τ I ss 0.4 ss I I 10 pA 10 2 20 0.2 20 ms 0 0 0 0 +++ - + - + - + - + - + - + + UBP UBP UBP UBP

Fig. 1. Effects of UBP-310, a GluK1-selective antagonist, on GluK1/GluK2 heteromers. (A) Cartoon representation of GluK1/GluK2 receptors with variable subunit composition. UBP-310 (blue circles) prevents glutamate from binding at GluK1 subunits (blue). (B) Typical current responses from GluK1 homomers (Left), GluK1/GluK2 heteromers (Middle), and GluK2 homomers (Right) to 3 mM Glu in the absence and presence of UBP-310 (concentrations as indicated). (C) Peak current reduction by UBP-310. (D) Relative steady-state currents in the absence and presence of UBP-310. (E) Effects on desensitization time constants

(τdes). (F) Additional data obtained in the continued presence of UBP-310 (dark blue). Measurements were performed by fast ligand application to outside-out patches from transfected HEK cells. The bars indicate mean values of normally distributed data from (n) different patches. Statistical testing was performed using paired t tests: **P ≤ 0.01; ***P ≤ 0.001; ns, not significant. More details are provided in SI Appendix, Figs. S1 and S2.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.2007471117 Pollok and Reiner Downloaded by guest on September 29, 2021 AMPA receptors remains unclear. It also raises the important The highest steady-state currents were observed for patches in question of how subunit-selective antagonists would affect the which UBP-310 binding caused the strongest current reduction, gating of heteromeric iGluRs, many of which are prevalent in the that is, in patches from cells that apparently expressed higher nervous system. levels of GluK1 than GluK2 (SI Appendix, Fig. S2C). Analyzing Here we show that UBP-310, a GluK1-selective antagonist, additional patches in the continued presence of UBP-310 showed reduces the desensitization of GluK1/GluK2 heteromers (for- that the steady-state currents can rise to >50% (mean Iss/peak = merly known as GluR5/GluR6) and fully abolishes the desensi- 29.7 ± 16.9%, n = 37 patches; Fig. 1F and SI Appendix,Fig.S2D). tization of GluK1/GluK5 heteromers (formerly GluR5/KA2). Overall, these data indicate that some receptor combinations with We find that similar effects are elicited by subunit-selective ag- mixed agonist/antagonist occupancies give nondesensitizing cur- onists and subunit-specific manipulations at heteromeric AMPA rents (SI Appendix, Note 1 and Fig. S8). receptors. Antagonist dissociation experiments provide addi- tional mechanistic insight into the behavior of homomeric re- UBP-310 Fully Suppresses Desensitization of GluK1/GluK5 Heteromers. ceptors. Overall, we find that partial ligand occupancy causes Endogenous kainate receptors often incorporate the high-affinity reduced desensitization, which may have direct implications for subunits GluK4 or GluK5 in addition to the low-affinity subunits, the use of subunit-selective ligands in neuroscience research and GluK1 to GluK3. The GluK5 subunit is widely expressed in the therapeutic applications. central nervous system (3, 19) and the incorporation of high- affinity subunits has profound consequences on receptor traffick- Results ing and gating (26, 56, 57). It has been shown that coexpression of UBP-310 Antagonizes GluK1 Homomers but Reduces Desensitization GluK2 and GluK5 subunits yields functional heteromers with of GluK1/GluK2 Heteromers. To test the influence of competitive defined 2:2 stoichiometry next to GluK2 homotetramers (58). subtype-specific antagonists on kainate receptor desensitization, GluK5 homotetramers remain nonfunctional and do not traffic to – we chose UBP-310. This derivative shows varying the membrane (56 59). binding affinities at different GluK subunits, with the highest af- Here we coexpressed GluK1 and GluK5 subunits, which – resulted in small but completely desensitizing currents with fast finity reported for GluK1 (52 54). I = ± We assessed the effect of UBP-310 on different kainate re- activation and desensitization kinetics (mean ss/peak 2.1 0.4%, mean τdesen = 0.7 ± 0.1 ms, n = 9 patches; Fig. 2 and SI ceptor combinations by performing fast glutamate perfusion ex- Appendix periments on outside-out patches from human embryonic kidney , Fig. S3), similar to GluK1 homomers. In the subse- (HEK) cells (Fig. 1A). Fast application of 3 mM glutamate to quent experiments, we lowered the UBP-310 concentration to μ μ SI PHARMACOLOGY GluK1 homotetramers yielded characteristic inward currents 1 M, since UBP-310 at 10 M also blocked GluK5 subunits ( B SI Appendix Appendix, Fig. S3B) (55). (Fig. 1 and ,Fig.S1). Fast activation and channel μ opening (rise time <0.4 ms; SI Appendix, Fig. S1A) was followed The addition of 1 M UBP-310 led to a strong but variable current reduction to 14% to 56% (n = 9 patches; Fig. 2 A and B), by rapid and almost complete desensitization (mean τ = 0.8 ± desen which indicates variable amounts of heteromeric GluK1/GluK5 0.1 ms; mean I = 1.0 ± 0.4%, n = 7patches;Fig.1B, steady-state/peak receptors next to GluK1 homomers. Most striking was the effect D, and E). Recovery from glutamate-induced desensitization oc- on desensitization. In the presence of 1 μM UBP-310, all cur- curred on the seconds timescale (τ = 2.0 s; SI Appendix,Fig. recov rents were completely nondesensitizing, that is, desensitization S1B), in line with previous reports (28). became fully blocked (mean I = 99.2 ± 4.3%, n = 9 patches; When we added 1 μM UBP-310 to the glutamate and wash ss/peak Fig. 2B), which was also confirmed in double-pulse experiments solutions during individual recordings, the glutamate-induced (SI Appendix, Fig. S3E). As a result, the use of a GluK1-specific currents were fully suppressed (Fig. 1 B and C), confirming antagonist caused a strong overall potentiation of the currents that UBP-310 acts as high-affinity antagonist at GluK1 (52, 53). originating from GluK1/GluK5 heteromers. After glutamate re- In contrast, the addition of UBP-310 had no significant effect on moval, the currents slowly deactivated (mean τdeac = 32.6 ± 7.3 the responses of GluK2 homotetramers, even when applied at a n = C μ B C μ ms, 9 patches; Fig. 2 ), which matches the glutamate deac- concentration of 10 M (Fig. 1 and ). However, at 100 M tivation kinetics that have been reported for GluK5 subunits (26). UBP-310 the responses of GluK2 homotetramers to 3 mM glu- SI Appendix E Overall, these data suggest that GluK1/GluK5 heteromers tamate were partially antagonized ( , Fig. S1 ), which yield half-maximal but nondesensitizing currents when glutamate is in line with previous reports (55). is prevented from binding at the GluK1 subunits. This behavior To test the influence of UBP-310 on heteromeric receptors, resembles the situation in GluK2/GluK5 heteromers, in which we coexpressed GluK1 and GluK2 subunits, which are expected agonist binding at either the two GluK2 subunits or the two GluK5 to form receptor complexes with varying stoichiometries of GluK1 subunits produces half-maximal activation but no desensitization and GluK2 subunits (20, 24). Indeed, currents from cotransfected (48) (SI Appendix, Note 1). cells showed desensitization kinetics intermediate between fast GluK1 and slow GluK2 desensitization (Fig. 1 B and E and SI Desensitization during Antagonist Dissociation from GluK1 and Appendix,Fig.S2). The addition of 10 μM UBP-310 caused a GluK1/GluK5 Receptors. Our experiments show that desensitiza- current reduction in all patches ranging from 14% to 93% (n = 17 tion is reduced when antagonists occupy only a part of the sub- patches; Fig. 1 B and C), which indicates that different amounts of units that are being present in heteromeric receptors. This suggests GluK1 subunits were present. Desensitization slowed in the that desensitization may also be reduced during antagonist disso- presence of UBP-310 (Fig. 1E), mainly because the remaining ciation while receptors are progressing through different occupancy currents were now dominated by GluK2-containing receptors. states (Fig. 3A). Most importantly, the addition of antagonist had a marked We first monitored the recurrence of glutamate-induced cur- effect on the amount of desensitization. The relative steady-state rents from GluK1 homotetramers after a saturating pulse of current, Iss/peak, increased from mean 1.9 ± 1.2% in the absence UBP-310 had been applied (Fig. 3B). The peak current recovered of UBP-310 to up to 32% in the presence of UBP-310 (mean slowly on the tens of seconds timescale with a pronounced lag I = ± n = B D ss/peak 16.8 8.3%, 17 patches; Fig. 1 and ). Double- phase (t1/2 = ∼35 s, n = 6 patches; Fig. 3C), as expected for re- pulse experiments show that these steady-state currents do not ceptors that are transitioning through states that yield no or little desensitize further, which indicates that they originate from activation. Similar kinetics were also reported for UBP-310 disso- stable receptor subpopulations that do not desensitize (SI Ap- ciation from desensitization-blocked GluK1 receptors (52). How- pendix, Fig. S2B). ever, a key observation is that we did not observe any significant

Pollok and Reiner PNAS Latest Articles | 3of8 Downloaded by guest on September 29, 2021 A GluK1/GluK5 can be summarized in a branched kinetic scheme (Fig. 3A). The implications of a more detailed version of this model are described 1 μM UBP-310 SI Appendix Note 2 3 mM Glu in , . If dissociation proceeded equally fast from all four subunits (koff = k′off), then all possible occupancy states would become significantly populated during UBP-310 dissociation from GluK1 homotetramers (SI Appendix, Fig. S10A). For statistical reasons, τdes= 0.8 ms τdeac= 31.6 ms path I would be slightly favored over path II (2:1). This model would reproduce the observed lag phase if we assume that re-

Iss/I peak= 93 % ceptors with one agonist bound do not yield current and that receptors with two, three, and four agonists bound contribute increasingly more current (SI Appendix, Note 1 and Fig. S8B). AUPB/A Glu= 0.28 −1 Least-squares fitting yields koff = k′off = 0.023 s (SI Appendix, B 5 pA 5 pA Fig. S4 ); however, this model also predicts significant steady- state currents (SI Appendix, Note 2), in contrast to our 5 ms 40 ms experimental observations. To explain this discrepancy, we explored alternative models. B (9) (9) C (9) (9) For instance, if cooperativity is at play, then dissociation of the second antagonist, k′ , may be faster than dissociation of the 1.2 *** 120 *** 1.0 50 off first antagonist from this dimer, koff. In this case, path II would SI Appendix 1.0 100 become increasingly favored over path I ( , Fig. 0.8 40 S10B). In addition, receptors with one antagonist (three ago- 0.8 80 nists) bound would then accumulate to a lesser extent. 0.6 30

(%) Evidence for the second scenario is provided by our data in- (ms)

(rel.) 0.6 60 (ms) dicating that UBP-310 dissociation from GluK1 subunits is much / I des

deac 20 peak 0.4 faster in the context of GluK1/GluK5 heteromers than in GluK1 τ I τ ss peak 0.4 I 40 homotetramers (Fig. 3). If we assume that GluK1/GluK5 het- 0.2 20 0.2 10 eromers incorporate two GluK1 subunits, as is the case in GluK2/GluK5 heteromers (48, 58) (SI Appendix, Note 1), then a −1 0 0 0 0 fit of the corresponding model gives k′off = 0.28 s (Fig. 3E and + - + - + SI Appendix, Fig. S4C). The observed lag phase suggests that UBP UBP UBP UBP receptors with one subunit blocked by UBP-310 (three agonists bound) produce partly nondesensitizing currents (SI Appendix, Fig. 2. The antagonist UBP-310 abolishes desensitization of GluK1/GluK5 Note 2 C C heteromers. (A) Typical glutamate responses after coexpression of GluK1 and Figs. S4 and S10 ). k′ = −1 and GluK5 subunits before (Left) and after (Right) the addition of 1 μM UBP- Once we incorporate off 0.28 s to describe UBP-310 310. (B) UBP-310 reduced the current (I ≤56%) and completely blocked dissociation from GluK1 homotetramers (Fig. 3C), we obtain peak − = ∼ τ k = 1 ∼ desensitization (Iss/Ipeak 99%). (C) Desensitization time constants ( des)in off 0.011 s ,a 25-fold difference in dissociation rate con- τ the absence of UBP-310 and deactivation time constants ( deac) in the pres- stants (k′off/koff). This readily explains why nondesensitizing or ence of UBP-310. Data are from n = 9 patches; bars indicate mean values. partially desensitizing configurations, namely path I and recep- Statistical testing was performed with paired t tests: ***P ≤ 0.001. See also SI tors with three bound agonists, do not significantly accumulate Appendix, Fig. S3. during antagonist dissociation (SI Appendix, Fig. S10D), in agreement with our experimental observations (Fig. 3 B and C).

steady-state currents during recovery from UBP-310 block (Iss/ The Effect of Subunit-Selective Antagonists Is Mirrored by peak ≤5%, n = 6patches;Fig.3C). Apparently, occupancy states Subunit-Selective Agonists. We found that desensitization is re- that produce nondesensitizing currents are not rate-limiting and do duced or fully abolished when a part of the subunits is occupied by thus not accumulate during dissociation. No significant changes in UBP-310. This inhibiting effect on desensitization could be spe- current rise time and desensitization kinetics were observed (SI cific to UBP-310 or could be due to reduced agonist occupancy. Appendix,Fig.S4A). To test the latter hypothesis, we investigated responses from For comparison, we measured glutamate-induced currents dur- heteromeric receptors using subunit-selective agonists as well as ing UBP-310 dissociation from GluK1/GluK5 heteromers (Fig. binding-impaired subunits. 3D). Binding of UBP-310 at the GluK1 subunits resulted in non- First, we used the agonist 5-iodowillardiine (5-IW), which is desensitizing glutamate currents, as shown previously (Fig. 2). On known to activate GluK1 and GluK5 but not GluK2 subunits (12, dissociation, also the GluK1 subunits became available for gluta- 24, 51). When we applied 100 μM 5-IW after coexpressing mate binding, and the current desensitized (Fig. 3D). The current GluK1 and GluK2 subunits, we obtained a similar outcome as ceased within seconds, showing a minor lag phase (t1/2 = ∼4.3 s, n = was seen in the presence of UBP-310: a part of the 5-IW current 3patches;Fig.3E). Thus, the loss of nondesensitizing configura- desensitized rapidly, but substantial steady-state currents remained tions in GluK1/GluK5 heteromers during UBP-310 dissociation is (mean Iss/peak = 15.6 ± 6.3%, n = 9 patches; Fig. 4A and SI Ap- considerably faster than the peak current recovery of GluK1 pendix,Fig.S5). This is in contrast to the full desensitization in- homotetramers. duced by 5-IW binding at all four subunits, as observed for GluK1 We analyzed our findings with a simplified model (Fig. 3A and homomers (SI Appendix, Fig. S5A) and GluK1/GluK5 heteromers SI Appendix Note 2 , and Fig. S9). Since iGluR tetramers as- (mean Iss/peak = 2.0 ± 1.3%, n = 6 patches; Fig. 4A and SI Appendix, semble with pseudo-twofold symmetry (36; but see ref. 60 for a Fig. S5B). recent exception) we assumed that UBP-310 dissociation starts The range of steady-state currents observed on 5-IW binding from a symmetric configuration. Different configurations can be to GluK1/GluK2 heteromers (5% to 25%; Fig. 4A) likely reflects distinguished during antagonist dissociation, however. Most obvi- heterogeneous receptor compositions, as has been described in a ously, two antagonists (agonists) can be bound at different LBD similar experiment on GluK1(Q)/GluK2(R) heteromers (24). In dimers (path I) or at the same LBD dimer (path II). The situation contrast, 5-IW binding to the GluK5 subunits in GluK2/GluK5

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.2007471117 Pollok and Reiner Downloaded by guest on September 29, 2021 GluK1 A path I C n=6

2 k off 2 k´off

4 k off k´off (%)

k´ 2 k peak off off I path II -1 koff= 0.011 s B GluK1 1 µM UBP-310 3 mM Glu

pre 5 s 20 s 45 s 100 s (%) peak I

/ ss I

4 pA 5 ms time (s)

GluK1/GluK5 GluK1/GluK5 D E n=3 1 µM UBP-310 3 mM Glu 3 mM Glu (40 s)

pre 80 ms -1 k´off = 0.28 s (%) ss I

τdes= 0.7 ms τdeac= 26.8 ms 10 pA 5 ms 20 ms 5 s time (s)

Fig. 3. Glutamate-induced currents during UBP-310 dissociation. (A) Kinetic scheme showing the different occupancy states that can become populated during antagonist dissociation. Two antagonists may be bound at different LBD dimers (path I) or at the same LBD dimer (path II). More details are provided in SI Appendix, Note 2.(B) Glutamate responses during UBP-310 dissociation from GluK1 homotetramers. Test pulses of 3 mM glutamate were applied before PHARMACOLOGY and at 5-s intervals after applying a pulse of 1 μM UBP-310. (C) Peak currents (Top) and steady-state currents (Bottom) normalized to the peak currents before −1 −1 UBP-310 application (mean ± SD, n = 6 patches). Fitting the kinetic scheme in A with k′off = 0.28 s (see below) yields koff = 0.011 s (red line). (D, Left) Glutamate responses of GluK1/GluK5 heteromers. UBP-310 binding at the GluK1 subunits blocks glutamate-induced desensitization. (D, Right) After UBP-310 dissociation, the GluK1 subunits become available for glutamate binding, and the steady-state current slowly desensitizes. (E) The disappearance of the −1 steady-state current (average current of three patches) shows a clear lag phase. Fitting of a three-state kinetic scheme yields a k′off = 0.28 s (red line). Details of the model are provided in SI Appendix, Note 2, and details on the fits are provided in SI Appendix, Fig. S4.

heteromers, which assemble in defined stoichiometries (58, 61), glutamate (mean Iss/peak = 3.6 ± 1.7%, n = 6; SI Appendix, Fig. results in completely nondesensitizing currents (48). Our findings S6B) or application of 4-MeGlu at GluA2(L671T) homotetramers are also supported by observations reported for ATPA, another (mean Iss/peak = 2.2 ± 1.4%, n = 10 patches; Fig. 4C). The latter GluK1- and GluK5-selective agonist (20). experiment confirms that the reduced desensitization does not Similarly, when we coexpressed GluK2(wt) and a binding- result from a low ligand efficacy of 4-MeGlu. impaired GluK2 subunit, GluK2(T690V) (59), we again observed Similarly, coexpression of GluA2(wt) and the low-affinity incomplete desensitization withsteady-statecurrentsupto15% variant GluA2(T501A) (SI Appendix, Fig. S7) (42) resulted in (mean Iss/peak = 5.2 ± 4.3%, n = 10 patches; Fig. 4B), in contrast to significantly higher steady-state currents on glutamate applica- the pronounced desensitization of GluK2(wt) homotetramers tion (mean Iss/peak = 7.5 ± 4.1%, n = 8 patches) compared with I = ± n = (mean Iss/peak = 1.3 ± 0.8%, n = 8 patches; Fig. 1). Taken together, GluA2(wt) homotetramers (mean ss/peak 3.4 1.7%, 26 these experiments confirm that occupancies can patches; Fig. 4D). Thus, we conclude that AMPA receptors be- result in incomplete desensitization of various kainate receptor have similarly to kainate receptors, with partial agonist occu- types. pancy resulting in partial desensitization.

Partial Agonist Occupancy Also Reduces Desensitization of Heteromeric Discussion AMPA Receptors. We next tested whether subunit-specific manip- Heteromeric iGluRs are widely expressed in the central nervous ulations would also affect AMPA receptor desensitization. For system, but little is known about how subunit-specific antagonists this, we artificially modified the GluA2 ligand selectivity by and agonists affect their activation and desensitization properties. substituting an side chain in the binding pocket (leu- In the first part of our study, we relied on UBP-310, a competitive cine 671 to ; L671T). The L671T substitution is known antagonist with reported selectivity for several kainate receptor to increase kainate binding and efficacy at GluA2 receptors (62), subunits (52, 53) that is being increasingly used in neuroscience and it has been suggested that leucine 671 obstructs binding of the research (e.g., refs. 55, 65). Our fast-ligand application experi- kainate receptor agonist (2S,4R)-4-methylglutamic acid (4-MeGlu; ments confirm high selectivity for GluK1 receptors over GluK2 SYM 2081) (63, 64). Indeed, we found that GluA2(L671T) receptors and, to a lesser degree, over GluK5-containing receptors homomers were rapidly activated and desensitized by 200 μM (Figs.1and2andSI Appendix,Figs.S1–S3), which is in line with 4-MeGlu, a concentration that does not elicit currents at GluA2(wt) ligand-binding assays and work on desensitization blocked recep- homotetramers (SI Appendix,Fig.S6). tors (52–54). However, in our hands, this specificity was not high Strikingly, when we applied 4-MeGlu after coexpressing enough to clearly differentiate between GluK2 and GluK5 sub- GluA2(L671T) and GluA2(wt) subunits, we observed steady- units (SI Appendix,Fig.S3B; but see ref. 55). state currents of up to 13% (mean Iss/peak = 6.9 ± 2.5%, n = 11 Most importantly, our observations show that by acting as a patches), significantly higher than those seen on application of subunit-specific antagonist, UBP-310 reduces the glutamate-induced

Pollok and Reiner PNAS Latest Articles | 5of8 Downloaded by guest on September 29, 2021 A GluK1/GluK2 B GluK2 T690V/wt The extent of desensitization block depends on receptor composition and thus on the relative expression levels and as- 100 μM 5-IW 1 mM Glu (9) (6) (10) (8) sembly preferences of the respective subunits. Steady-state cur- 30 *** 20 * rents from GluK1/GluK2-coexpressing cells and GluK2 or GluA2 pseudoheteromers were variable and mostly rather small, > 15 although in some patches steady-state currents reached 50% 20 (Fig. 1F and SI Appendix, Note 1). A different behavior is seen (%) (%) I /I = 10.5 % 10 for kainate receptor complexes that incorporate the high-affinity

ss peak peak 6.7 % I / I / subunit GluK5. Previous work with tethered photoswitchable ss ss peak I 10 I 5 ligands and selective agonists has provided detailed insight into 4 pA 5 pA the activation and desensitization of GluK2/GluK5 heteromers, 20 ms 0 20 ms 0 which assemble with defined 2:2 stoichiometry (58) and where K1/2 K1/5 690V/wt wt liganding of either the two GluK2 or the two GluK5 subunits GluA2 L671T/wt GluA2 T501A/wt provides half-maximal activation but no desensitization (48). C D Here we obtained similar results for GluK1/GluK5 heteromers 200 μM 4-MeGlu 3 mM Glu using UBP-310 to block glutamate from binding at GluK1 sub- (11) (10) (8) (26) units (Figs. 2 and 3 D and E). This indicates that GluK1/GluK5 20 *** 20 * heteromers also have a strong preference for assembling into nondesensitizing configurations, possibly with 2:2 stoichiometry 15 15 8.8 % 13.5 % (SI Appendix, Note 1). Studies with isolated amino-terminal domains (%) (%) (ATDs) suggest a strong preference for heterodimeric GluK1/ 10 10 peak I I GluK5 assemblies, similar to those of GluK2/GluK5 (61, 66). / / ss peak ss I I In another set of experiments, we addressed whether non- 5 5 desensitizing states are also populated during antagonist disso- 5 pA 20 pA 0 ciation (Fig. 3). We found that UBP-310 dissociates on slower 20 ms 0 20 ms 671T/wt 671T 501A/wt wt timescales than typical antagonists (31, 41), making it well suited for these experiments. In the case of Fig. 4. Subunit-specific agonists and binding-impaired subunits reduce de- GluK1/GluK5 heteromers, the UBP-310 block was lost within sensitization of heteromeric kainate and AMPA receptors. (A) Application of seconds, as demonstrated by the concomitant loss of steady-state the GluK1-specific agonist 5-IW causes significant steady-state currents of current (Fig. 3 D and E). The kinetics shows a lag phase, indi- GluK1/GluK2 heteromers (green) but not of GluK1/GluK5 heteromers (gray). cating that receptors with three bound agonists also contribute to (B) Incorporation of binding-impaired GluK2(T690V) subunits significantly SI Appendix Note 2 increases glutamate-induced steady-state currents compared with homo- this nondesensitizing current ( , ). In contrast, meric GluK2(wt) receptors (gray). (C) GluA2(L671T) is activated by 4-MeGlu, UBP-310 dissociation from GluK1 homotetramers was slower, resulting in small steady-state currents that are typical for GluA2 (gray). The occurring on the tens of seconds timescale, during which we did 4-MeGlu-induced steady-state currents are significantly higher in heteromers not detect any significant steady-state currents (Fig. 3 B and C). with GluA2(wt) (green). (D) Incorporation of binding-impaired GluA2(T501A) These observations can be readily explained if we assume that subunits into GluA2 receptors significantly increases glutamate-induced antagonist dissociation is not equally fast at all four receptor steady-state currents. The bars indicate mean values, and n indicates the subunits. These differences could be due to binding cooperativity ’ number of different patches. Statistical testing was performed with Welch s and/or positional differences within the tetramers (SI Appendix, t tests: *P ≤ 0.05, ***P ≤ 0.001. More details on the experiment and the Note 2 controls are provided in SI Appendix,Figs.S5–S7. ). Our data also show heterogeneity in the receptor con- figurations with two bound agonists: some configurations pro- duce nondesensitizing currents, while other configurations with two bound agonists produce either no or fully desensitizing re- desensitization of heteromeric GluK1/GluK2 receptors (Fig. 1) and sponses. This situation can be illustrated by a branched kinetic fully abolishes the desensitization of GluK1/GluK5 heteromers scheme (Fig. 3A). (Fig. 2). We further show that desensitization is similarly reduced by More generally, our experiments show that ligand occupancy is subunit-selective agonists or the incorporation of binding-impaired an important determinant of AMPA and kainate receptor de- subunits, as demonstrated by 5-IW binding to GluK1/GluK2 het- sensitization, in addition to ligand efficacy (67, 68) and structural eromers and GluK2/GluK2(T690V) pseudoheteromers, respectively rearrangements of the LBD dimer interface (42–44, 69). The (Fig. 4 A and B). This suggests that the inhibitory effects on de- prevailing view that one agonist-occupied subunit is sufficient to sensitization result from incomplete agonist occupancy. Thus, we induce efficient desensitization of the tetrameric receptors (26, expect that reduced desensitization will be generally observed when 47) had already been challenged by work on kainate receptors purely competitive antagonists block agonist binding at only a part incorporating the high-affinity subunits GluK4 and GluK5 (48–50) of the four subunits. and here is also disproven for other types of kainate receptors, as We next asked whether AMPA receptor desensitization would well as GluA2 AMPA receptor pseudoheteromers. Further ex- be similarly affected. AMPA receptors are the dominant post- perimental work is needed to address the role of binding coop- synaptic receptor species at excitatory and are fre- erativity and interdimer coupling (34), the behavior of different quently heteromers (4, 21–23). Since only few subunit-selective types of AMPA receptor heteromers, and the contribution of AMPA receptor agonists are currently available (1, 18), we auxiliary subunits. Besides the mechanistic implications of our engineered an AMPA receptor subunit, GluA2(L671T), that al- findings, we show that partial receptor occupancies can have sig- lows for 4-MeGlu binding and also used a binding-impaired sub- nificant practical consequences, for instance, when subunit-selective unit, GluA2(T501A), to form pseudoheteromeric receptors with antagonists are used to treat heteromeric receptor populations. GluA2(wt) subunits (Fig. 4 C and D). We found that, as in kainate The desensitization block that can arise from using subunit- receptors, partial agonist occupancies caused partial desensitiza- selective antagonists may be small and variable, but the physio- tion. Thus, one occupied subunit is not sufficient to efficiently logical consequences could be large. Heteromeric AMPA re- trigger full receptor desensitization, in contrast to the predictions ceptors play an important role in the nervous system (4, 21–23), of models typically used to describe iGluR gating (26, 47) (SI and recent work suggests that their assembly occurs in a biased, Appendix, Note 1). nonrandom fashion (22, 25, 70). In this case, the formation of

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.2007471117 Pollok and Reiner Downloaded by guest on September 29, 2021 nondesensitizing configurations may be favored, similar to in therapeutic applications. These findings should motivate more GluK5-containing kainate receptor heteromers in which prefer- detailed investigations analyzing the effects of subunit-selective ential assembly (58, 61, 66) yields nondesensitizing configura- ligands and modulators on iGluR function. tions while high-affinity glutamate binding is preserved (Fig. 2). The impact on synaptic function may be profound if iGluRs Materials and Methods maintain nondesensitizing currents in the presence of falling or Currents were measured by fast piezo-driven glutamate application to low basal glutamate concentrations. The use of subunit-specific outside-out patches from HEK cells transiently expressing the indicated antagonists may prolong synaptic activations or cause unwanted kainate and AMPA receptor heteromers. Voltage-clamp measurements were − tonic activations. Moreover, since blocking of desensitization performed at 70 mV using the indicated ligands. Details of the constructs, measurements, and data analysis are provided in SI Appendix, Materials circumvents the need for slow recovery, postsynaptic short-term and Methods. depression also may be affected. In summary, we show that competitive antagonists with high Data Availability. All study data are included in the main text and SI Appendix. subunit-selectivity can cause unintended effects at heteromeric iGluRs. Depending on the receptor composition, they reduce ACKNOWLEDGMENTS. We thank P. H. Seeburg (MPI Heidelberg), Y. Stern- desensitization. This is important to keep in mind when subunit- Bach (Hebrew University), K. M. Partin (Colorado State University), L. Chen selective antagonists are used in neurophysiological experiments, (Stanford University), and E.Y.I. for iGluR expression plasmids and Hendrik Margis, Laura Hönig, and Jeannette Gebel for mutagenesis and/or initial for example, when UBP-310 or its derivative ACET is used to whole- characterization of receptor variants. We also thank Nadine Hube dissect metabotropic kainate receptor signaling (55, 65, 71). Fur- for excellent technical assistance and Adela Dudic for discussion. This project thermore, subunit-selective ligands may cause new adverse effects was funded by a grant from the NRW-Rückkehrprogramm (to A.R.).

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