This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glutamate receptors are the major excitatory neurotransmitter receptors in vertebrate brain and are involved in a variety of normal and pathological neuronal functions. These proteins function by binding glutamate in an extracellular domain and opening an intrinsic ion channel that allows cations to flow in and out of the neuron. Drugs targeted to glutamate receptors may have considerable potential for treating such diverse disorders as epilepsy, amyotrophic lateral sclerosis, and ischemic brain damage. We are studying two important glutamate receptors (GluR2 and GluR3), using X-ray crystallography and NMR and ESR spectroscopy to understand the structure and dynamics and to compare the results with the function of the protein measured using single channel recording (measurement of ion conductance across the cell membrane). The structural work is done on the extracellular ligand-binding domains of the proteins (GluR2 S1S2 and GluR3 S1S2), which are a soluble constructs derived from the full-length proteins. The proteins have a bilobed structure with the binding site for glutamate and derivatives at the interface between the two lobes. For GluR2, previous work has suggested that the degree to which the lobes close upon binding of ligand may relate to the function of the protein. Our initial work with NMR spectroscopy and our recent crystal structures, suggest that the relationship between structure and function may be more complicated, involving protein flexibility. Obtaining additional structures, under conditions that reveal the range of possible motions, is essential for understanding the functional consequences of agonist binding. In collaboration with the Sondermann laboratory, we have obtained the structures of GluR2 S1S2 bound to several new ligands (1.5 to 1.7 angstrom resolution). The time requested in this cycle will be used to determine structures of GluR2 S1S2 with three new ligands under two crystallization conditions that we suspect will provide an indication of the range of possible motions of the protein. We also have crystals for GluR3 S1S2, the structure of which has not yet been determined. This protein is also available bound to at three different ligands. In addition the importance of understanding its crucial function in the central nervous system, the rationale for expanding these studies to the GluR3 subtype is that our functional studies of GluR3 have advanced considerably in recent years, and the hope of understanding in detail the link between structural changes in the binding domain to the conductance of ions through the cell membrane is very promising with this subtype. We have determined the conditions and have grown all of the crystals to be used in these studies. As noted above, the crystals that we have obtained from GluR2 diffract to high resolution (1.5 to 1.7 angstroms), and we anticipate similar results from the new crystals that we have obtained.
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