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 Unpublished data from the host lab confirm in principle such a regulatory role.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.

Normal brain functions such as memory formation, cognition, and learning rely on the proper communication of nerve cells via synapses. AMPA-type glutamate receptors (AMPARs) are located in the postsynaptic site, where they open their ion-pore upon the neurotransmitter glutamate binding. Thereby, AMPARs mediate the majority of excitatory signaling in the mammalian brain. Altered AMPARs are implicated in neurological disorders such as epilepsy, schizophrenia, and Alzheimer's disease. This project aimed to unravel the molecular details underlying receptor activation and modulation by auxiliary subunits termed TARP proteins. In a first approach, we monitored the receptor movement from the resting state (no ligand bound and closed pore) to the glutamate bound active state (pore open) towards the desensitized state (glutamate is still bound but the pore is already closed) in functional experiments. Therefore, we trapped the receptor in its different activation states by introducing redox-sensitive or metal-trapping mutations. In combination with structural studies, using X-ray crystallography, we found that the receptors' ligand-binding domain layer is highly dynamic. Solely when agonists, like glutamate bind, the ligand-binding domain layer is stabilized enough to allow ion channel opening. In a second approach, we investigated the modulatory role of TARPs on receptor kinetics. In previous studies, TARPs were shown to directly associate with AMPA receptors and thereby influencing their opening times and desensitization rates. However, the exact regions responsible for these receptor modulations were not known. Using in silico structural modeling, we predicted areas that might harbor the modulatory properties. These hypotheses were tested in electrophysiological experiments. For the first time, we could show that TARPs interfere at a receptor site that transmits the signal from the ligand-binding event towards the pore. The findings of this project contributed greatly to our understanding of AMPAR receptor activation and modulation and highlighted possible ways to interfere with AMPAR signaling in disease models. Ideally, our findings might inspire future drug development approaches.