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

Abstract

Ionotropic glutamate receptors (iGluRs) are key molecules for synaptic signaling in the central nervous system, which makes them promising drug targets. Intensive efforts are being devoted to the development of subunit-selective ligands, which should enable more precise pharmacologic interventions while limiting the effects on overall neuronal circuit function. However, many AMPA and kainate receptor complexes in vivo are heteromers composed of different subunits. Despite their importance, little is known about how subunit-selective ligands affect the gating of heteromeric iGluRs, namely their activation and desensitization properties. Using fast ligand application experiments, we studied the effects of competitive antagonists that block glutamate from binding at part of the four subunits. We found that UBP-310, a kainate receptor antagonist with high selectivity for GluK1 subunits, reduces the desensitization of GluK1/GluK2 heteromers and fully abolishes the desensitization of GluK1/GluK5 heteromers. This effect is mirrored by subunit-selective agonists and heteromeric receptors that contain binding-impaired subunits, as we show for both kainate and GluA2 AMPA receptors. These findings are consistent with a model in which incomplete agonist occupancy at the four receptor subunits can provide activation without inducing desensitization. However, we did not detect significant steady-state currents during UBP-310 dissociation from GluK1 homotetramers, indicating that antagonist dissociation proceeds in a nonuniform and cooperativity-driven manner, which disfavors nondesensitizing occupancy states. Besides providing mechanistic insights, these results have direct implications for the use of subunit-selective antagonists in neuroscience research and envisioned therapeutic interventions.

Structural and compositional diversity in the kainate receptor family

“…Second, we did not functionally assay different di-, tri-, or tetra-heteromers. However, electro-physiological measurements of heterologously expressed di-heteromers containing GluK1–3 show properties varying from cell to cell, indicating the expression of variable receptor populations ( Cui and Mayer, 1999 ; Pollok and Reiner, 2020 ). Our results imply that the heteromers recorded in these experiments may have included a combination of 3:1, 2:2, and 1:3 stoichiometries.…”

“…KARs are tetrameric channels that assemble from five subunit types (GluK1–5). GluK1, GluK2, and GluK3 share ~75% identity ( Table S1 ) and can form functional homo-tetramers (homomers) on their own or can co-assemble with any other KAR subunit to form hetero-tetramers (heteromers) ( Cui and Mayer, 1999 ; Mulle et al, 2000 ; Pinheiro et al, 2007 ; Pollok and Reiner, 2020 ; Veran et al, 2012 ). In contrast to GluK1–3, GluK4 and GluK5 are obligate heteromer subunits, which must co-assemble into tetramers with GluK1–3 to function ( Herb et al, 1992 ; Werner et al, 1991 ).…”

“…However, our understanding of KAR composition and subunit compatibility has been largely driven by electro-physiological and biophysical studies investigating KAR homomers and KAR di-heteromers (i.e., tetramers composed of two different subunit types). Di-heteromers of GluK1, GluK2, or GluK3 form functional channels, but their stoichiometries are un-clear ( Cui and Mayer, 1999 ; Pollok and Reiner, 2020 ; Veran et al, 2012 ). GluK2/K5 di-heteromers are reported to harbor two copies of each subunit type (2:2 ratio) ( Khanra et al, 2021 ; Litwin et al, 2020 ; Reiner et al, 2012 ), although whether other stoichiometries are possible is not known.…”

sDarken: Next generation genetically encoded fluorescent sensors for serotonin

“…We next characterized the temporal characteristics of the 5-HT sensor variants. To achieve fast ligand application and removal, we used membrane patch fluorometry in conjunction with rapid, piezo-driven solution exchange 25 . Outside-out membrane patches from sDarken -expressing HEK cells were excised with a patch pipette and placed in front of a piezo-mounted double-barreled theta-glass pipette, which allows for submillisecond switching between a solution containing 5-HT and solution without 5-HT (Supplementary Fig.…”

Splicing and editing of ionotropic glutamate receptors: a comprehensive analysis based on human RNA-Seq data

“…NMDA receptors are obligatory heteromers of two GluN1 and two GluN2 (GluN2A-D) or GluN3 (GluN3A/B) subunits. Heteromer formation also appears to prevail within the AMPA and kainate receptor subfamilies, which allows for the integration of different functionalities within single receptor complexes [19][20][21].…”