lonotropic glutamate receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system and are implicated in a number of wide-ranging effects in the body. Glutamate binding to an extracellular domain initiates a series of conformation changes that ends in the formation of a cation selective transmembrane channel, which then closes due to desensitization of the receptor. The crystal structures of the isolated ligand binding domain of the AMPA subtype (LBD) gives knowledge into the structural changes in the LBD; however, these structures lack transmembrane segments, the primary functional part of the protein. To gain a more complete understanding of how agonist binding is coupled to channel activation and desensitization, it is necessary to study changes in the LBD in the presence of the transmembrane segments. This can be achieved by using fluorescence resonance energy transfer (FRET). To measure conformational changes modified AMPA receptor can be tagged at specific sites. Using this modified receptor, we propose to study the conformational changes by establishing distance changes between specific sites on the protein tagged with donor and acceptor fluorophores. The first specific aim is to establish whether cleft closure in the ligand binding domain is the primary control of receptor activation using various ligands and mutations at the L650 and Y450 sites, which induces a wide range of activations. The second specific aim is to determine the conformational changes associated with desensitization by testing the dimer interface hypothesis for desensitization, which is thought to be caused by the separation of the interface between two subunits in the glutamate receptor dimer to relieve stress on the transmembrane segments induced by cleft closure. Through the use of these techniques, ligands, and mutants, agonist binding coupled to channel activation and desensitization will be investigated in the full receptor. Regarding health issues, glutamate receptors are the primary mediators of fast excitatory synaptic transmission via ionotropic receptors and are also involved in more intricate neuronal processes, such as learning and memory. Glutamate-mediated excitotoxicity implicated in neuronal damage associated with various neurological insults and diseases, including ischemia, anoxia, stroke, hypoglycemia, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, lathyrisms, and Alzheimer's disease. Gaining a better understanding of structure-function correlations and determining the molecular basis of the receptor function is a key step towards designing and developing the next generation of drugs that can alter the function of this important receptor and hence aid in the treatment of the clinical conditions listed above. ? ? ?
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