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[Abstract]

Structural Basis for AMPA Receptor Activation, Deactivation and Ligand Selectivity

Anders Hogner

Ionotropic glutamate receptors (iGluRs) constitute a family of ligand-gated ion channels that are essential for mediation of fast synaptic transmission. The iGluRs are implicated in learning and memory and in neurologic and psychiatric diseases. They are divided into three classes based on sequence identity and their pharmacological response to the agonists 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartic acid (NMDA). The AMPA-preferring receptors are assembled from homo- or heteromeric combinations of the subunits GluR1-4, and are suggested to be tetramers. The heterogeneity is enhanced by alternative splicing and post-transcriptional RNA editing. The ligand binding core (S1S2), responsible for binding the neurotransmitter, is made up of two discontinuous extracellular domains.

In recent years, our understanding of activation and deactivation of iGluRs at a cellular level has substantially increased, however little is known about the molecular mechanisms behind these processes. Consequently, detailed structural studies of AMPA receptors are highly desirable for gaining a better understanding of the protein-ligand interactions and deeper knowledge of the functional mechanisms associated with iGluRs.

A breakthrough occurred with the determination of X-ray crystal structures of the ligand binding core of the AMPA receptor subunit GluR2 (GluR2-S1S2) alone and in complex with a series of ligands, by the group of Eric Gouaux, Columbia University. The structures revealed some fundamental details of ligand recognition and paved the way for a model describing the mechanisms of activation and deactivation of iGluRs.

The thesis describes expression, refolding, and purification of the GluR2-S1S2 construct; the protocol and construct were kindly provided by E. Gouaux. The protein was characterized by [3H]-AMPA binding assays and shown to display typical AMPA receptor pharmacology. The protein was co-crystallised with the three agonists: (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxyisoxazol-5-yl)propionic acid (Br-HIBO) and with one antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)isoxazol-4-yl]propionic acid (ATPO). The structures reveal: 1) different binding modes of the ligands; 2) key residues for ligand recognition; 3) a new mechanism for obtaining an antagonist effect; 4) the importance of the water molecule architecture; and 5) a consistent 2-fold symmetric dimer in the crystal structures. Furthermore, a point mutation was introduced yielding a GluR2-S1S2-Y702F construct which mimics the ligand-binding core of GluR3-S1S2. Subsequently structure determination of the two complexes S1S2-Y702F:Br-HIBO and S1S2-Y702F:ACPA identified the molecular mechanism behind the observed selectivity of Br-HIBO for GluR1 over GluR3, and extended the understanding of protein ligand interactions beyond GluR2. A computational study was undertaken, which highlighted the importance of an interdomain interaction within the ligand binding site of iGluRs.

Agonist binding to S1S2 induces domain closure compared to the open binding cleft of the apo state. By performing functional experiments on homomeric GluR2 receptors expressed in oocytes a clear correlation became apparent, between the degree of domain closure and the efficacy of the agonists.

Taken together, the series of molecular snapshots reported here is a valuable step towards a better understanding of the molecular mechanisms behind activation and deactivation of iGluRs. Moreover, the structures provide a solid platform and new challenges for the ongoing research directed towards designing novel AMPA receptor ligands.

 

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