第18回細胞生理学セミナー（先端薬科学特論・単位認定）

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain “layer” of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs. In native tissues, AMPA receptors are predominantly organized as hetero-tetramers, assembled from two different isoforms. GluA1/A2 receptors represent the dominant isoform combination in CA1 pyramidal neurons of the hippocampus, but their stoichiometry and spatial arrangement is unknown. We have purified heteromeric GluA1/A2 receptors and used GluA2-specific antibody fragments (Fabs) to distinguish between GluA1 and GluA2 isoforms by fluorescence-size exclusion chromatography and in single particle cryo-EM experiments. Using 3D reconstructions of receptor/Fab complexes of homomeric GluA2 and heteromeric GluA1/A2 at ~10 Å resolution in conjunction with cysteine cross-linking experiments, we demonstrate that GluA1/A2 receptors heteromerize with a preferred 2:2 stoichiometry and a distinct spatial arrangement of 1-2-1-2.

References: Dürr et al., 2014, Cell / Chen, Dürr & Gouaux, 2014, Science