Ionotropic glutamate receptors control a wide variety of normal neuronal processes including learning and memory. Activation of these neurotransmitter receptors is involved in a number of neurodegenerative diseases, notably stroke and epilepsy. Analysis of the transmembrane topology has led to the realization that each subunit is made of a series of modules. The module that binds glutamate can be produced in bacteria as a soluble protein (S1S2 domain) and its structure has been determined. The S1S2 domain of AMPA receptors binds agonists and antagonists with approximately the same affinity as the intact receptor and serves as an excellent system for studying the binding domain. In the previous granting period, backbone and sidechain dynamics were studied with a series of partial agonists bound to GluR2 S1S2 using NMR spectroscopy. These measurements, with studies of solution structure, new crystal structures, and isothermal titration calorimetry (ITC), have shed light on the mechanism of agonist and antagonist binding and the relationship between dynamics of the binding domain and channel gating. Based on the results, the relationship between dynamics and channel gating can be approached on a more quantitative level, using mutagenesis designed to modify large-scale lobe motions and backbone motions that correlate with agonist efficacy. In particular, the mechanism by which conformational changes in the S1S2 domain lead to channel activation will be investigated. Because glutamate receptors are thought to function as dimers of dimers, an appropriate model for the activated channel would be the dimer of the S1S2 domain. The protein is monomeric below 6 mM, but the L483Y mutation results in dimeric protein at much lower concentrations. This mutation blocks desensitization in the intact protein, and desensitization is correlated with the dissociation of the dimer interface. Preliminary studies indicate that dimerization leads to changes in the agonist binding site, and the goal will be to determine the effects of dimerization on dynamics and thermodynamics of agonist and antagonist binding. Differences between the dimeric and monomeric states should provide clues as to how the protein changes upon desensitization. Finally, allosteric activators will be studied. These drugs enhance cognition and are being tested in neurological disorders such as Alzheimer's disease. NMR spectroscopy, X-ray crystallography, radioligand binding, and ITC will be used to determine the mechanisms of binding and important interactions with the receptor for a series of activators with differing interactions with the binding domain. These studies will use a range of biophysical techniques with whole cell and patch clamp recording of receptor function to study the agonist and allosteric activator binding sites on the GluR2 AMPA receptor. The results will shed light on the correlation of structure, function and dynamics of an important glutamate receptor subunit and provide essential information for development of drugs that are antagonists or allosteric activators.
AMPA receptors mediate the majority of fast excitatory synaptic transmission in the central nervous system. Over-activity of these receptors has been implicated in contributing to the pathological effects of stroke and epilepsy, and enhancement of the activity of AMPA receptors has been shown to be beneficial in increasing cognition. For this reason, both agonists and activators of these receptors are likely to be very important therapeutically. Drugs targeted to specific subtypes and splice variants have the potential for more selective action with possibly fewer side effects. The goal of these studies is to understand the structure, function and dynamics of one of the most prevalent and medically relevant AMPA receptors (GluR2), and to provide the groundwork for the development of new therapeutic agents.
|Martinez, Madeline; Ahmed, Ahmed H; Loh, Adrienne P et al. (2014) Thermodynamics and mechanism of the interaction of willardiine partial agonists with a glutamate receptor: implications for drug development. Biochemistry 53:3790-5|
|Ptak, Christopher P; Hsieh, Ching-Lin; Weiland, Gregory A et al. (2014) Role of stoichiometry in the dimer-stabilizing effect of AMPA receptor allosteric modulators. ACS Chem Biol 9:128-33|
|Ahmed, Ahmed H; Ptak, Christopher P; Fenwick, Michael K et al. (2013) Dynamics of cleft closure of the GluA2 ligand-binding domain in the presence of full and partial agonists revealed by hydrogen-deuterium exchange. J Biol Chem 288:27658-66|
|Ahmed, Ahmed H; Hamada, Makoto; Shinada, Tetsuro et al. (2012) The structure of (-)-kaitocephalin bound to the ligand binding domain of the (S)-Î±-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/glutamate receptor, GluA2. J Biol Chem 287:41007-13|
|Ahmed, Ahmed H; Wang, Shu; Chuang, Huai-Hu et al. (2011) Mechanism of AMPA receptor activation by partial agonists: disulfide trapping of closed lobe conformations. J Biol Chem 286:35257-66|
|Fenwick, Michael K; Oswald, Robert E (2010) On the mechanisms of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor binding to glutamate and kainate. J Biol Chem 285:12334-43|
|Maltsev, Alexander S; Oswald, Robert E (2010) Hydrophobic side chain dynamics of a glutamate receptor ligand binding domain. J Biol Chem 285:10154-62|
|Ahmed, Ahmed H; Oswald, Robert E (2010) Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. J Med Chem 53:2197-203|
|Ahmed, Ahmed H; Ptak, Christopher P; Oswald, Robert E (2010) Molecular mechanism of flop selectivity and subsite recognition for an AMPA receptor allosteric modulator: structures of GluA2 and GluA3 in complexes with PEPA. Biochemistry 49:2843-50|
|Ahmed, Ahmed H; Wang, Qi; Sondermann, Holger et al. (2009) Structure of the S1S2 glutamate binding domain of GLuR3. Proteins 75:628-37|
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