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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Gerratana, Barbara
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University of Texas Health Science Center Houston
Schools of Medicine
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Rambhadran, Anu; Gonzalez, Jennifer; Jayaraman, Vasanthi (2011) Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor. J Biol Chem 286:16953-7
Wu, Mousheng; Tong, Shuilong; Gonzalez, Jennifer et al. (2011) Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing. Structure 19:1509-17
Landes, Christy F; Rambhadran, Anu; Taylor, J Nick et al. (2011) Structural landscape of isolated agonist-binding domains from single AMPA receptors. Nat Chem Biol 7:168-73
Rambhadran, Anu; Gonzalez, Jennifer; Jayaraman, Vasanthi (2010) Subunit arrangement in N-methyl-D-aspartate (NMDA) receptors. J Biol Chem 285:15296-301
Gonzalez, Jennifer; Du, Mei; Parameshwaran, Kodeeswaran et al. (2010) Role of dimer interface in activation and desensitization in AMPA receptors. Proc Natl Acad Sci U S A 107:9891-6
Du, Mei; Rambhadran, Anu; Jayaraman, Vasanthi (2009) Vibrational spectroscopic investigation of the ligand binding domain of kainate receptors. Protein Sci 18:1585-91
Gonzalez, Jennifer; Rambhadran, Anu; Du, Mei et al. (2008) LRET investigations of conformational changes in the ligand binding domain of a functional AMPA receptor. Biochemistry 47:10027-32
Liu, Zhenyu; Ramanoudjame, Gomathi; Liu, Deqian et al. (2008) Overexpression and functional characterization of the extracellular domain of the human alpha1 glycine receptor. Biochemistry 47:9803-10
Mankiewicz, Kimberly A; Rambhadran, Anu; Wathen, Lisa et al. (2008) Chemical interplay in the mechanism of partial agonist activation in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Biochemistry 47:398-404
Du, Mei; Rambhadran, Anu; Jayaraman, Vasanthi (2008) Luminescence resonance energy transfer investigation of conformational changes in the ligand binding domain of a kainate receptor. J Biol Chem 283:27074-8

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