Domain Architecture of a Calcium-Permeable AMPA Receptor in a Ligand-Free Conformation

Charles R Midgett,Avinash Gill,Dean R Madden

doi:10.3389/fnmol.2011.00056

Abstract

Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPA receptors, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation.

Highlights

Glutamate is the most common excitatory neurotransmitter in the brain, estimated to be responsible for signaling at 80% of cortical synapses (Braitenberg and Schüz, 1998)
As with all mammalian ionotropic glutamate receptors (iGluRs), AMPA receptor (AMPAR) subunits have a modular domain architecture composed of an extracellular N-terminal domain (NTD), a bilobate ligand-binding domain (LBD), a transmembrane domain (TMD), and a cytoplasmic C-terminus (Wo and Oswald, 1995)
To obtain experimental constraints on the domain architecture of the ligand-free state, we obtained full-length GluA2-Q receptors with fluorescent protein tags inserted at positions adjacent to the NTD and known to be compatible with the assembly of functional receptors (Braithwaite et al, 2002; Sheridan et al, 2006)

Summary

Introduction

Glutamate is the most common excitatory neurotransmitter in the brain, estimated to be responsible for signaling at 80% of cortical synapses (Braitenberg and Schüz, 1998) It activates ionotropic glutamate receptors (iGluRs), a family of ligand-gated ion channels that assemble as functional tetramers. IGluRs have been implicated in synaptic plasticity, a key neurological component of learning and memory (Derkach et al, 2007), and play important roles in pathological processes, those involving the excitotoxicity associated with excess Ca2+ influx (Liu and Zukin, 2007) They participate in a variety of non-neuronal autocrine and paracrine signaling processes (Hinoi et al, 2004). As with all mammalian iGluRs, AMPAR subunits have a modular domain architecture composed of an extracellular N-terminal domain (NTD), a bilobate ligand-binding domain (LBD), a transmembrane domain (TMD), and a cytoplasmic C-terminus (Wo and Oswald, 1995)

Methods

Results

Conclusion