Ionotropic glutamate receptors (iGluRs) are membrane proteins that 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 biochemical and electrophysiological experiments to define the mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. This important 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 structural studies on full length iGluRs. Glutamate receptor ion channels are multidomain membrane proteins which assemble as 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 one quarter of the mass of an intact receptor. A major challenge in the field is to establish techniques for the expression and purification of mg quantities of full length iGluRs. Our approach uses fluorescence size exclusion chromatography (FSEC) as a semi-automated approach for screening large numbers of constructs, and for testing effects of different ligands and detergent and lipid combination on the stability of oligomeric assemblies. For baculovirus infected insect cell cultures grown on the 12 liter scale we can routinely obtain between 0.5 to 2 mg of the kainate receptor GluK2 with the yield and purity varying with solubilization techniques. Detergent solubilization of whole cells gives higher yields that membrane preparations, but with reduced purity. In addition to pursuing crystallization for GluK2 we have also started a collaboration with Sriram Subramaniam using single particle cryo-electron tomography to determine the structures, at 25 resolution, of full-length GluK2 kainate receptors trapped in resting and desensitized states. The current EM analysis used full length kainate receptors with native glycosylation in conditions that maximized receptor stability. Membranes were solubilized in a DDM-CHS mixture that thermostabilizes GPCRs, and most likely other membrane proteins, due to a sterol mediated ordering effect on the DDM hydrocarbon tail, such that the central core of the resulting micelle resembles a membrane-like bilayer. In addition, compared to the use of the apo state and glutamate complexes in prior studies on AMPA receptors, GluK2 was trapped in either resting or desensitized states using high affinity ligands; the much greater stability of the desensitized state of GluK2 compared to AMPA receptors also likely contributed to improved conformational homogeneity of the desensitized state. The resulting GluK2 structures differed substantially from those reported in prior EM studies on AMPA receptors, but remarkably the resting state was nearly identical to the GluA2 crystal structure, with an upright position of the ATD dimers, as opposed to the tilted ATDs observed in previous negative-stain EM work. The GluK2 desensitized state was also strikingly different from structures observed in prior EM studies on AMPA receptors. The ATDs in the GluK2 desensitized state structure retain a nearly identical structure to the resting state differing greatly from AMPA receptor Type II structures, some of which require extremely large ATD dimer movements that are difficult to reconcile with subunit crossover observed in the GluA2 crystal structure, and the short length of the linkers connecting the ATD and LBD. We believe that these differences result from improved specimen preparation and imaging analysis that is possible using cryo-electron tomography on vitrified preparaptions in which receptors are trapped in a thin layer of ice, compared to negative stain procedures and image reconstruction techniques. Structural studies on allosteric modulation of GluK3 kainate receptors by zinc Kainate receptors (KARs) play a key role in the regulation of synaptic networks. The Mulle lab in Bordeaux found that zinc, a cation released at a subset of glutamatergic synapses, potentiates glutamate currents mediated by homomeric and heteromeric KARs containing GluK3 at 10-100 &#956;M concentrations, whereas it inhibits other KAR subtypes. Potentiation of GluK3 currents is mainly due to reduced desensitization, as shown by kinetic analysis and modeling. Mutation and crystallographic analyses performed at NIH revealed that a specific zinc binding site is formed at the base of the Ligand Binding Domain (LBD) dimer interface by a GluK3-specific aspartate (Asp759), together with two conserved residues, His762 and Asp730, the latter located on the partner subunit. Experiments with mutant tetrameric GluK2/3 assemblies reveals that these are likely assembled as pairs of heterodimeric LBDs. In combination this analysis reveals that zinc binding stabilizes the labile GluK3 dimer interface, slows desensitization and potentiates currents, providing a new mechanism for KAR potentiation at glutamatergic synapses. Structural analysis of novel iGluR ligand binding domains A glutamate receptor sequence recently discovered in a primitive eukaryote, Adineta vaga, exhibits characteristics similar to both the prokaryotic GluR0 and different classes of eukaryotic iGluRs. Large scale expression of the AvGluR1 ligand binding domain was achieved, allowing radioligand binding assays and structural analysis to be performed with the goal of examining the molecular mechanisms for glutamate receptor evolution. In ongoing work it has been established that AvGluR1 binds alanine and serine with high affinity, as well as glutamate and aspartate. Crystal structures have been solved for each of these constructs and reveal novel unexpected ligand and ion interactions that differ from those found in eukaryotic glutamate receptors NMDA Receptor Glycine and Glutamate Ligand-Binding Domains In a collaboration with Albert Lau at Johns Hopkins University we are undertaking an analysis of NMDA receptor activation using a combined structural (NIH) and computational (Lau) approach. Central to the Venus fly trap model for iGluR activation is the underlying assumption that the open-cleft apo state is a stable conformational entity, and that the ligand binding domain (LBD) rarely undergoes cleft closure in the absence of agonists. Support for this model was obtained from calculation of free-energy landscapes for the GluA2 LBD, which revealed distinct and well defined energy minima for the apo and agonist bound states, the conformation of which agreed well with that for GluA2 LBD crystal structures. Experimental structures for the apo states of other iGluR receptor families have not been solved, and their conformational stability remains unknown. We have now crystallized the NMDA receptor GluN1 and GluN3A LBDs in their apo states and are calculating free energy landscapes.