Probing Intersubunit Interfaces in AMPA-subtype Ionotropic Glutamate Receptors.

Appu K Singh,Maria V Yelshanskaya,Alexander I Sobolevsky,Kei Saotome

doi:10.1038/srep19082

Appu K Singh, Maria V Yelshanskaya + Show 2 more

Open Access

https://doi.org/10.1038/srep19082

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Journal: Scientific reports	Publication Date: Jan 7, 2016
Citations: 18	License type: CC BY 4.0

Affiliation: Columbia University

Abstract

AMPA subtype ionotropic glutamate receptors (iGluRs) mediate the majority of fast neurotransmission across excitatory synapses in the central nervous system. Each AMPA receptor is composed of four multi-domain subunits that are organized into layers of two amino-terminal domain (ATD) dimers, two ligand-binding domain (LBD) dimers, transmembrane domains and carboxy-terminal domains. We introduced cysteine substitutions at the intersubunit interfaces of AMPA receptor subunit GluA2 and confirmed substituted cysteine crosslink formation by SDS-PAGE. The functional consequence of intersubunit crosslinks was assessed by recording GluA2-mediated currents in reducing and non-reducing conditions. Strong redox-dependent changes in GluA2-mediated currents were observed for cysteine substitutions at the LBD dimer-dimer interface but not at the ATD dimer-dimer interface. We conclude that during gating, LBD dimers undergo significant relative displacement, while ATD dimers either maintain their relative positioning, or their relative displacement has no appreciable effect on AMPA receptor function.

Highlights

Ionotropic glutamate receptors are a family of tetrameric ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the central nervous system and are implicated in numerous devastating neurological diseases[1,2,3]
We introduced nine mutations at or near the intersubunit interfaces of rat GluA2i AMPA-subtype ionotropic glutamate receptors (iGluRs) (Fig. 1A): four cysteine substitutions at the amino-terminal domain (ATD) dimer-dimer interface (I209C, I211C, G212C and V215C; Fig. 1B), three cysteine substitutions at the ligand-binding domain (LBD) dimer-dimer interface (K663C, I664C and A665C; Fig. 1C), one cysteine substitution at the LBD-TMD linker region (R628C; Fig. 1D) and one cysteine substitution in the ion channel (A621C; Fig. 1D)
We probed the functional effect of crosslinking ATD and LBD interdimer interfaces located along the axis of overall two-fold rotational symmetry (Fig. 1)

Summary

Introduction

Ionotropic glutamate receptors (iGluRs) are a family of tetrameric ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the central nervous system and are implicated in numerous devastating neurological diseases[1,2,3]. While the family includes members with distinct biophysical and pharmacological properties, such as NMDA, AMPA and kainate receptors, each iGluR subunit has a conserved modular design that is comprised of ATD, LBD, ion channel and CTD. Domain interaction by virtue of intersubunit interfaces plays a crucial role in iGluR assembly[4,5,6,7,8]. The ATD dimer-dimer interface appears prominent in different isolated ATD17–21 and full-length iGluR crystal structures[5,16,22,23], consistent with a purely structural role in non-NMDA receptor assembly. We probed the relative positions of iGluR domains during gating by evaluating the functional consequence of introducing cysteine crosslinks at the interdomain interfaces along the axis of overall two-fold rotational symmetry (Fig. 1)

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