Vibrational Spectroscopic Investigations of Glutamate Receptor

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DESCRIPTION (provided by applicant): The specific molecular interactions between glutamate and the glutamate receptor are central to the allosteric regulation of function in this membrane bound protein, which is main excitatory neurotransmitter receptor in the mammalian central nervous system. Recent spectroscopic investigations of the isolated ligand-binding domain of the glutamate receptor in the apo and ligand bound forms provide a first view of such interactions. What is now needed is a more detailed understanding of the role of these chemical interactions in mediating the sequence of conformational changes that ultimately regulate ion flow. We propose to address this goal using two strategies. First, using a panel of mutants that exhibit a wide range of activities we will identify changes in specific ligand protein interactions using vibrational spectroscopy and correlate these to changes in the cleft closure conformational change in the ligand binding domain using fluorescence resonance energy transfer measurements. The changes thus identified in the ligand binding domain will then be contextualized in terms of changes in receptor function that will be studied by electrophysiological measurements. Secondly, we will monitor the kinetic events in the ligand binding domain and correlate these to the functional consequences in the receptor, i.e., activation and desensitization. For such a correlation, a panel of mutants that are known to alter one or more of the kinetic steps in the ligand binding domain will be used and the specific effects on the structural changes in the ligand binding domain as well as corresponding changes in function will be investigated. These equilibrium and kinetic investigations will provide comprehensive understanding of the changes at the molecular level that drive the changes in the large conformational changes in the ligand binding domain and eventually control the functional changes of the ion channel. More importantly, such correlations will aid in bridging the gap between studies on the isolated ligand-binding domain and the behavior of the intact receptor, and provide a basis for rational design of drugs aimed at modulating the function of this important protein which is known to play an important role in diverse neuropathologies, including epilepsy and ischemia.