Subunit-selective iGluR antagonists can potentiate heteromeric receptor 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 pain (LY5454694) (17). In the case of AMPA receptors, synaptic signaling in the central nervous system, which makes which have more closely related ligand-binding domains (LBDs), them promising drug targets. Intensive efforts are being devoted some moderately selective agonists have been reported, but to the development of subunit-selective ligands, 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 kainate receptor complexes in vivo are heteromers composed receptor subtypes and to the brain 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. receptor antagonist 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 agonist 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 ion channel | medicinal chemistry | non-NMDA receptor | Significance receptor modulation Ionotropic glutamate receptors (iGluRs) are a diverse family of lutamate is an important neurotransmitter 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 genes (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, epilepsy, mood disorders, and addiction, 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 ischemia (LY377770) (13), anxiety (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