Structural and Functional Insights into GluK3-kainate Receptor Desensitization and Recovery

Abstract

GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic transmission. For a better understanding of structural changes that underlie receptor functions, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. While the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a resting state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using structures as a guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. The mutational analysis also identified putative N-glycan interacting residues. Our results provide a molecular framework for understanding gating properties unique to GluK3 and exploring the role of N-linked glycosylation in their modulation.

Architecture and structural dynamics of the heteromeric GluK2/K5 kainate receptor

“…This study shows that the LBD layer of desensitized GluK2/K5 is organized with pseudo-fourfold symmetry and with the four LBDs positioned such that their G helices are in a staggered arrangement near the central axis of the receptor ( Figure 4E). This LBD configuration is similar to that observed in desensitized GluK2 ( Figure 4H) (Meyerson et al, 2016) and GluK3 (Kumari et al, 2019) which suggests the arrangement is a hallmark of desensitization in the KAR family. The mechanistic role of this LBD arrangement cannot yet be fully understood because no active state KAR structure is available.…”

“…The small re-entrant helices (M2) were not resolved, likely because of conformational mobility. The TMD structure ( Figure 3A) has a 'trapezoidal' shape and a square base ( Figure 3B) which is seen in other members of the iGluR family (Herguedas et al, 2019;Karakas and Furukawa, 2014;Kumari et al, 2019;Lee et al, 2014;Lü et al, 2017;Meyerson et al, 2016;Sobolevsky et al, 2009). The antagonistbound structure is expected to feature a closed ion channel so to understand how GluK2/K5 em restricts cation flow we visualized the pore profile using HOLE (Smart et al, 1996) (Figure 3C-F).…”

“…The channel closes again if L-Glu is rapidly removed (deactivation) or if L-Glu exposure is sustained (desensitization) ( Barberis et al, 2008 ). Recent structures have shown how GluK2 and GluK3 homomers are organized in the absence of L-Glu while bound to competitive antagonists and after the receptors have desensitized ( Kumari et al, 2019 ; Meyerson et al, 2014a ; Meyerson et al, 2016 ; Schauder et al, 2013 ). The desensitized structures revealed that KAR homomers feature a ‘desensitization ring’ motif which is proposed to facilitate channel closure and account for their slow recovery from desensitization ( Meyerson et al, 2016 ).…”

“…We conclude with structural analysis to answer how antagonist-bound and desensitized GluK2/K5 states can both maintain a closed cation channel despite such different LBD arrangements. Critically, this work provides an expanded foundation for understanding differences and similarities among KAR ( Kumari et al, 2019 ; Meyerson et al, 2014a ; Meyerson et al, 2016 ), AMPAR ( Herguedas et al, 2019 ; Zhao et al, 2019 ), NMDAR ( Karakas and Furukawa, 2014 ; Lee et al, 2014 ; Lü et al, 2017 ), and recently characterized ionotropic orphan delta (GluD) receptor structures ( Burada et al, 2020a ; Burada et al, 2020b ).…”

The Architecture of GluD2 Ionotropic Delta Glutamate Receptor Elucidated by cryo-EM

“…However, we cannot rule out the influence of grid preparation conditions and air-water interface interactions in observation of this unique asymmetric arrangement of the two-extracellular arms. Moreover in the absence of domain swapping, the conformational freedom for the movement of the two dimer arms is substantially higher as seen in the GluD1 receptors (Burada et al, 2020) and unlike that in AMPA (Dürr et al, 2014;Twomey et al, 2017) and kainate receptors (Meyerson et al, 2016;Kumari et al, 2019) and we could have trapped a physiologically relevant conformational intermediate. It's also important to note that this conformation may not be compatible with trans-synaptic, tripartite complex formed between GluD2, cerebelin, and neurexin which is essential for maintaining the synaptic integrity of PF-PC synapses as this would limit the large-scale motions of the two extracellular arms.…”