The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate.

Marek Ladislav,Martin Horak,Ladislav Vyklicky,Jan Krusek,Ales Balik,Jiri Cerny

doi:10.3389/fnmol.2018.00113

Abstract

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the central nervous system, underlie the induction of synaptic plasticity, and their malfunction is associated with human diseases. Native NMDARs are tetramers composed of two obligatory GluN1 subunits and various combinations of GluN2A-D or, more rarely, GluN3A-B subunits. Each subunit consists of an amino-terminal, ligand-binding, transmembrane and carboxyl-terminal domain. The ligand-binding and transmembrane domains are interconnected via polypeptide chains (linkers). Upon glutamate and glycine binding, these receptors undergo a series of conformational changes leading to the opening of the Ca2+-permeable ion channel. Here we report that different deletions and mutations of amino acids in the M3-S2 linkers of the GluN1 and GluN2B subunits lead to constitutively open channels. Irrespective of whether alterations were introduced in the GluN1 or the GluN2B subunit, application of glutamate or glycine promoted receptor channel activity; however, responses induced by the GluN1 agonist glycine were larger, on average, than those induced by glutamate. We observed the most prominent effect when residues GluN1(L657) and GluN2B(I655) were deleted or altered to glycine. In parallel, molecular modeling revealed that two interacting pairs of residues, the LILI motif (GluN1(L657) and GluN2B(I655)), form a functional unit with the TTTT ring (GluN1(T648) and GluN2B(T647)), described earlier to control NMDAR channel gating. These results provide new insight into the structural organization and functional interplay of the LILI and the TTTT ring during the course of NMDAR channel opening and closing.

Highlights

Excitatory signal transduction in the mammalian brain is primarily mediated by glutamate-activated ionotropic receptors represented by α-amino-3-hydroxyl-5-methyl-4isoxazole-propionate (AMPA), kainate and N-methyl-Daspartate receptor (NMDAR) subtypes (Dingledine et al, 1999; Traynelis et al, 2010)
Simple mechanistic view suggests that conformational changes in the ligand-binding domain (LBD) result in the movement of M3-S2 linkers followed by a centrifugal displacement of GluN1 and GluN2B M3 helices and channel opening
Our results indicate that an extracellular LILI motif which is formed by the M3-S2 linkers is an important element that connects conformational changes at the level of the LBD induced by agonist binding and unbinding to the NMDAR channel opening and closing

Summary

Introduction

Excitatory signal transduction in the mammalian brain is primarily mediated by glutamate-activated ionotropic receptors (iGluRs) represented by α-amino-3-hydroxyl-5-methyl-4isoxazole-propionate (AMPA), kainate and N-methyl-Daspartate receptor (NMDAR) subtypes (Dingledine et al, 1999; Traynelis et al, 2010). In addition to NMDAR-mediated postsynaptic Ca2+ current, unconventional presynaptic NMDARs were identified at several types of synapses, where they modulate presynaptic neurotransmitter release (Dore et al, 2017; Bouvier et al, 2018). Part of this NMDAR action is Ca2+-insensitive metabotropic signal transduction where a conformation change in the cytoplasmatic part of receptor activates an intracellular signaling pathway. Agonist binding evokes movement at the level of the S2 lobe (Furukawa et al, 2005; Yao et al, 2013) and it was suggested that agonist-induced conformation changes impact three polypeptide linkers connecting to the TMD forming the ion channel (Karakas and Furukawa, 2014). Based on the analysis of the available crystal structures of NMDAR and of isolated LBD structures, the clamshell-like displacement of the S2 with respect to the S1 is relatively small and insufficient to explain the magnitude of the displacement at the level of the TMD

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