Glutamate receptors play an important role in formal brain function. They are implicated in long term potentiation and long term depression, processes thought to underlie memory and learning. Glutamate receptors have also been implicated in many pathophysiological conditions affecting central nervous system function such as epilepsy, some neurodegenerative diseases, and neuronal cell death during ischemia and hypoglycemia. Thus, despite their importance in synaptic function, very little is known about their overall structure. The long term objectives of the proposed study are to determine which parts of the sequence of glutamate receptor subunits are exposed to the extracellular or cytoplasmic surface of the synaptic membrane (topology) and to determine the number of each kind of subunit which assembles to form recombinant glutamate receptors (stoichiometry). The topology of glutamate receptor subunits will be determined by first introducing reporter epitopes at various locations within a subunit, then expressing these engineered subunits in Xenopus oocytes, and finally determining the intracellular or extracellular location of the epitopes, with respect to the surface membrane of oocytes, by the binding of reporter monoclonal antibodies. Determining the topology of these receptor subunits should begin to reveal domains that might interact with intracellular structural components of glutaminergic synapses, analogous to those found for other ion channels, such as the 43K protein for the muscle-type acetylcholine receptor, and gephyrin for the glycine receptor. Extracellular domains that might contribute to the binding sites of established endogenous neurotransmitters such as glycine and L-glutamate and other potential neurotransmitters or modulators such as polyamines and arachidonic acid also might be identified. The subunit stoichiometry of a recombinant NMDA receptor will be determined. NMDA receptor subunits tagged with epitopes will be metabolically labeled with [35S]methionine and expressed as a heteromer with an invariant stoichiometry from subunit cRNAs in oocytes. The subunit stoichiometry will be deduced by determining the ratio of radiolabeled subunits of fully assembled receptors isolated from oocyte surface membranes. Elucidating the subunit stoichiometry is a first step towards determining the complete structure of NMDA receptors, as well as the contributions of individual subunit domains to the formation of ligand binding sites and the ion channel pore. Extracellular and intracellular domains identified by these studies then can be targeted by site-directed mutagenesis to better understand their functional roles. By carefully comparing the properties of recombinant NMDA receptors of known subunit stoichiometries, with those of native NMDA receptors from neurons expressing the same combinations of subunits, it might be possible to correlate the stoichiometries of some recombinant receptors to those of specific subtypes of native receptors.
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