Ionotropic glutamate receptors (iGluRs) are membrane proteins which act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. The 7 gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits which assemble to form 3 major functional families named after the ligands which were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate and NMDA. Because of their essential role in normal brain function and development, and increasing evidence that dysfunction of iGluR activity mediates multiple neurological and psychiatric diseases, as well as damage during stroke, a substantial effort in the Laboratory of Cellular and Molecular Neurophysiology is directed towards analysis of iGluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework in which to design electrophysiological and biochemical experiments to define the mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. This information will allow the development of subtype selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine which plays a key role in brain function. Expression and crystallization studies on the amino terminal domain of iGluRs Glutamate receptor ion channels are multidomain membrane proteins which assemble of tetramers of molecular weight approximately 440 kD. Numerous crystal structures have been solved for the ligand binding domains which have a molecular weight of approximately 30 kD per subunit, approximately of the mass of an intact receptor. Extensive trials with bacterial expression systems, which with one exception, have been used for all published ligand binding domain structures, failed to produce monodisperse soluble protein for other iGluR domains. The amino terminal domain (ATD) is an important structural target because it controls subtype selective assembly in native iGluRs, limiting assembly to members of the same functional family. Protein expression at levels sufficient for structural biology in mammalian cells is much more difficult than expression in E.coli but has the advantages that multiple check points select for correctly folded proteins, and add sugars and other post translational modifications required for normal function. Although a variety of cell biological and biochemical techniques are required to subsequently trim the sugar chains, in order to obtain proteins which crystallize and diffract to high resolution, and the yields are lower than for prokaryotic expression, currently this is the only approach likely to succeed for studies of the ATD. In ongoing work the ATDs from several iGluR subtypes have been screened for expression in mammalian cells. Crystallization trials have been performed using a nano liter pipetting robot. The structure of the GluK2 (GluR6) ATD revealed dimer and tetramer assemblies.Using the crystal structure for the GluK2 kainate receptor ATD as a guide, we performed cysteine mutant cross linking experiments in full length tetrameric GluK2 to establish how the ATD packs in a dimer of dimers assembly. A similar approach, using a full length AMPA receptor GluA2 crystal structure as a guide, was used to design cysteine mutant cross links for the GluK2 LBD dimer of dimers assembly. The formation of cross linked tetramers in full length GluK2 by combinations of ATD and LBD mutants which individually produce only cross linked dimers, suggests that subunits in the ATD and LBD layers swap dimer partners. Functional studies reveal that cross linking either the ATD or LBD inhibits activation of GluK2 and that, in the LBD, cross links within and between dimers have different effects. These results establish that kainate and AMPA receptors have a conserved extracellular architecture, and provide insight into the role of individual dimer assemblies in activation of ion channel gating. Kainate receptors are further classified into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptor families based on their affinity for the neurotoxin kainic acid. These two families share 42% sequence identity for the intact receptor but only 28% sequence identity at the level of ATD. We have determined for the first time high-resolution crystal structures for the GluK3 and GluK5 ATDs, both of which crystallize as dimers, albeit with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5 the R2 domain dimer assembly is similar to that reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-3. Our analysis also reveals that these non-NMDA receptor ATDs undergo only moderate variations in domain closure, of up to 10 in contrast to the 50 movement reported for the NMDA receptor GluN2A and GluN2B subunits. This restricted domain movement in non-NMDA receptor ATDs seems to result from both extensive intra-domain contacts, and from their assembly as dimers which interact at the R2 domains. Our results provide the first insights into the structure and function for GluK4-5, the least understood family of iGluRs. Expression studies are on going with the goal of obtaining a full length crystal structure of the ion channel assembly
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