An Ampa Receptor Unique Asp-Lys Salt-Bridge has a Role in Channel Function

Abstract

Several of the many steps in the mechanism of activation of tetrameric GluA receptor-channels have been explored in a variety of published structural and functional studies. The main findings from them are that 1) Agonist binding induces closure of the L1/L2 of the isolated ligand-binding domain (LBD) that is less for partial agonists than for glutamate. 2) Crystal structures of GluA LBDs bound to full agonists manifest a 180o rotation around an Asp-Ser transpeptide bond in a flexible part of L2 that leads to the formation of cross-interface H-bonds also hypothesized to play a role in receptor activation. 3) Channel conductance increases with increasing agonist concentration, consistent with multiple LBDs in the tetramer contributing to full channel activation. Here we show that the Asp of the Asp-Ser pair also makes an electrostatic contact with a Lys residue in helix-F, and that disrupting this salt-bridge in GluA3flip receptors with point mutations at D655 and K660 decreased agonist potency by 2-10 fold. In D655A where it was examined, this occurred without altering intrinsic receptor affinity for agonists in ligand binding assays or CNQX by Schild analysis. The D655A mutation also increased the efficacy of kainate relative to glutamate, and the isolated GluA-D655A LDB bound to kainate was 3° more closed than in wildtype. Pharmacological studies of the wildtype and mutant receptors expressed in human embryonic kidney cells employed whole cell patch recordings and structure data was from crystals of isolated wildtype and mutant LBDs bound to full agonists and kainate. Taken together, these studies showed that breaking the D655-K660 contact altered channel gating, indicating that tethering a flexible part of L2 to helix-F may contribute to the rapid activation of GluA receptors.