Structure-function analysis of glutamate receptor activity

The ionotropic glutamate receptors (iGluRs) are ligand gated ion channels that mediate the majority of excitatory transmission in the mammalian brain. iGluRs play an essential role in almost all aspects of nervous system function and development as learning, memory formation and homeostasis. Dysfunctional iGluRs are implicated in devastating neurodegenerative disorders, such as Alzheimers and Parkinsons disease, psychiatric conditions as schizophrenia and depression and acute disorders like stroke and epilepsy. The iGluR family is subdivided in three major subtypes, AMPA, kainate and NMDA receptors. Whereas these subfamilies exhibit diverse pharmacological and kinetic properties they share common structural characteristics. iGluRs assemble of four subunits, each comprising a large extracellular amino-terminal domain (ATD), involved in subtype specific receptor assembling, trafficking and modulation; a ligand binding domain (LBD) essential for agonist or antagonist binding, a ion channel forming transmembrane domain (TMD) and a carboxyl-terminal domain (CTD) engaging in synaptic localization, mobility and regulation. Once glutamate gets released into the synaptic space it binds to the LBD of postsynaptic iGluRs. Subsequently, the receptor undergoes several distinct rearrangements to open the channel and to desensitize within milliseconds. Although numerous structural studies on isolated LBDs and a structure of the full length receptor were recently published, we are lacking detailed knowledge of the active and desensitized state in the physiological tetrameric context. The first aim of this proposal is designed to provide this essential information in order to build a comprehensive model of iGluR activation. Therefore, we will introduce cysteine or histidine mutations at specific sites in the LBD, which will trap distinct activation states for crystallization. In a second step these trapping mutations will be subjected to electrophysiological studies to confirm our trapping hypothesis and to provide the physiological relevance. Recent studies showed that ATDs can regulate gating in NMDA receptors. However, the molecular mechanism of ATD regulation remains unclear. To date it is unknown if ATDs also have a regulatory function in AMPA and kainate receptor function as it was recently shown for NMDA receptors. Unpublished data from the host lab confirm in principle such a regulatory role. In a second proposed aim we will further investigate the regulatory capacity of ATDs in AMPA receptor gating by focusing on the linker region between ATDs and LBDs. We will use a similar trapping approach as described in aim 1 to structurally and functionally characterize ATDs. Investigating the ATD-LBD coupling is essential to understand if it is possible to control receptor activity through ligands that bind to the ATD. Results from this proposal will enhance our knowledge of glutamate receptor activation and modulation in normal brain function as well as in disease.

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