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Molecular architecture of heteromeric kainate receptor complexes

Periodic Reporting for period 1 - KARhet (Molecular architecture of heteromeric kainate receptor complexes)

Reporting period: 2018-06-18 to 2020-06-17

The mammalian nervous system is a complex network containing many types of cells, each playing a specific role in the transmission of information. The main excitatory neurotransmitter is L-glutamate, mediating fast neurotransmission through ionotropic glutamate receptors (iGluRs), which are divided into three sub-families: NMDA, AMPA and KA receptors. While AMPA and NMDA receptors have a prominent and well-characterised role in fast excitatory transmission, KA receptors (KARs) are modulators of synaptic circuits.

In the native context, KARs rely on heteromeric assembly and binding partners for their proper function. Indeed, the most abundant KARs are thought to be formed of the two subunit subtypes, GluK2 and GluK5. Heteromeric assembly of these subunits endows synaptic KARs with unique biophysical and pharmacological properties. Moreover, these receptors associate with auxiliary proteins at both pre- and postsynaptic membranes and this influences their trafficking, clustering and functional properties; detailed structural information regarding these complexes still remains to be determined.

KARs have been associated with disorders of the central nervous system such as mood disorders and temporal-lobe epilepsy. Given their role as neuronal circuit modulators, they represent an important potential pharmacological target, especially compared to AMPA and NMDA receptors, whose roles in synaptic transmission and plasticity are crucial. However, this requires a deeper understanding of synaptic KAR assemblies.

The overall aim of this project was to investigate the rules of assembly of KAR complexes in structural terms, including specific interactions between subunits in a heteromeric complex, as well as those between the receptors and their binding partners. Using single-particle cryo-electron microscopy (cryo-EM), this project provides a first and preliminary insight into the rules of assembly of a synaptic KAR complex.
The structural study of the KAR complex was very challenging. Several constructs were designed and screened for optimal protein purification. To improve protein yield, cell lines stably expressing KAR subunits and auxiliary proteins were created. A preliminary low-resolution cryo-EM map of the KAR in complex with an auxiliary protein helped in the design of constructs to be used in combination with X-ray crystallography to help solve high-resolution structures; these experiments are still ongoing.
The structural study of the KAR complex is still ongoing. Once the project is completed, the structural information gained will give valuable insight into the interactions between KAR subunits and their binding partners, which will ultimately help in our understanding of synaptic KAR complexes and opening avenues for potential therapeutic targets.
Preliminary cryo-EM map of the GluK2 homomer in a nanodisc