Two Signaling Modes Are Better than One: Flux-Independent Signaling by Ionotropic Glutamate Receptors Is Coming of Age

Abstract

Glutamate is the major excitatory neurotransmitter in the central nervous system. Glutamatergic transmission can be mediated by ionotropic glutamate receptors (iGluRs), which mediate rapid synaptic depolarization that can be associated with Ca²⁺ entry and activity-dependent change in the strength of synaptic transmission, as well as by metabotropic glutamate receptors (mGluRs), which mediate slower postsynaptic responses through the recruitment of second messenger systems. A wealth of evidence reported over the last three decades has shown that this dogmatic subdivision between iGluRs and mGluRs may not reflect the actual physiological signaling mode of the iGluRs, i.e., α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA) receptors (AMPAR), kainate receptors (KARs), and N-methyl-D-aspartate (NMDA) receptors (NMDARs). Herein, we review the evidence available supporting the notion that the canonical iGluRs can recruit flux-independent signaling pathways not only in neurons, but also in brain astrocytes and cerebrovascular endothelial cells. Understanding the signaling versatility of iGluRs can exert a profound impact on our understanding of glutamatergic synapses. Furthermore, it may shed light on novel neuroprotective strategies against brain disorders.

Keywords: AMPA receptors; NMDA receptors; flux-independent signaling; glutamate; ionotropic glutamate receptors; kainate receptors; non-canonical signaling.

Figure 1

Flux-independent signaling pathways activated by AMPARs in neurons. AMPARs can signal in a flux-independent manner to recruit several signaling pathways. AMPARs can interact with G_i/o proteins to either inhibit voltage-gated Ca_V2.1 channels or activate voltage-gated Na⁺ channels (Na_V). Extracellular Na⁺ entry through Na_V channels can promote mitochondrial Ca²⁺ release through the mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). In addition, AMPARs can signal through G_i/o proteins to accelerate vesicle recycling or induce BDNF gene expression through ERK activation, which is mediated by the interaction between AMPARs and the tyrosine kinase, Lyn. Alternately, AMPARs can activate the ERK phosphorylation cascade to promote cell survival via the G_i/o protein-dependent recruitment of the PI3K/Akt pathway. Finally, AMPARs can also signal through G_i/o proteins to inhibit the expression of the Arc gene.

Figure 2

Flux-independent signaling by KARs modulates the intracellular Cl⁻ concentration ([Cl⁻]_i) and the reversal potential for GABA (E_GABA). (A) Pharmacological stimulation of postsynaptic KARs with KA (green circle) reduces the [Cl⁻]_i in hippocampal CA3 pyramidal neurons. Upon permissive cPKC-dependent phosphorylation, KARs promote the recycling of the K⁺–Cl⁻ cotransporter 2 (KCC2) from Rab11-containing vesicles to the PM; the tight interaction between KAR and KCC2 stimulates K⁺ and Cl⁻ efflux into the extracellular milieu, thereby reducing the [Cl⁻]_i and increasing extracellular Cl⁻ influx through GABA_ARs. (B) Synaptically released glutamate (red circle) stimulates flux-independent signaling by KARs, which engage cPKC to phosphorylate extrasynaptic GABA_ARs and thereby increase extracellular Cl⁻ influx in hippocampal CA1 pyramidal neurons.

Figure 3

Flux-independent signaling by KARs modulates neuronal excitability and synaptic plasticity. Flux-independent signaling by KAR can increase neuronal excitability by inhibiting I_sAHP and I_mAHP via G_i/o proteins and cPKC. Additionally, KARs can stimulate the exocytosis of AMPARs from Rab1-containing vesicles on the PM of dendritic spines. KARs trigger a signaling cascade involving G_i/o proteins and phospholipase C (PLC). PLC, in turn, synthesizes the two intracellular second messengers: DAG, which activates cPKC, and InsP₃, which induces ER Ca²⁺ release through InsP₃ receptors (InsP₃Rs). The combined effect of cPKC and Ca²⁺ release results in the recruitment of Rab11 endosomes to the PM, thereby increasing the surface expression of AMPARs.

Figure 4

Flux-independent signaling by NMDARs in neurons. (A) Agonist and co-agonist binding leads to the dephosphorylation of GluN1 Y837 and GluN2A Y842, resulting in clathrin-dependent internalization via the adaptor protein AP-2. High concentrations (10 µM) of glycine alone increase the interaction with AP-2, thereby priming NMDARs for dynamin-dependent endocytosis upon agonist binding. (B) Agonist and co-agonist binding to GluN1-containing NMDARs can stimulate the p38 MAPK in a flux-independent manner to promote AMPAR endocytosis, resulting in Ca²⁺-independent LTD induction. (C) Flux-independent signaling by NMDARs regulates spine morphology. In the presence of weak Ca²⁺ entry, NMDARs promote dendritic spine shrinkage by triggering a signaling pathway that requires interaction between nNOS and NOS1P, involving p38 MAPK, MK2, and cofilin, which promotes actin depolymerization. This signaling pathway is supported by mTORC1, which is likely to drive new protein synthesis. In the presence of strong Ca²⁺ influx (highlighted in red), the Ca²⁺-dependent recruitment of CaMKII leads to dendritic spine growth via inducing actin polymerization. (D) Flux-independent signaling by postsynaptic NMDARs regulates glutamate release by stimulating Src kinase to activate pannexin 1 (PANX1) channels, which clear synaptic anandamide (AEA) and prevent AEA-induced activation of presynaptic TRPV1 channels. This causes a reduction in [Ca²⁺]_i at the presynaptic terminal and therefore decreases Ca²⁺-dependent glutamate release. By contrast, upon agonist and co-agonist binding, presynaptic NMDARs promote glutamate release via the JNK2-dependent signaling pathway. In all the panels, the red circle indicates the agonist, whereas the blue circle indicates the co-agonist. ↑: increase.

Figure 5

Flux-independent signaling by NMDARs in brain disorders. (A) Flux-independent signaling by NMDARs leads to neuronal excitotoxicity upon massive glutamatergic stimulation. NMDARs trigger InsP₃-induced ER Ca²⁺ release, which inhibits the EF-2 protein and interferes with protein synthesis. Furthermore, NMDARs can stimulate Ca²⁺ entry through PANX1 channels via Src-dependent phosphorylation of PANX1. Excessive Ca²⁺ entry leads to mitochondrial Ca²⁺ overload, mPTP opening, and apoptosis. Finally, agonist binding to GluN2B-containing NMDARs can remove the p85 regulatory subunit from the catalytic domain of PI3K, thereby inducing PI3K-dependent NADPH oxidase-2 (NOX2) activation. NOX2 can also be activated via Ca²⁺ entry through ionotropic NMDARs and lead to cytotoxic superoxide production. The red circle indicates the agonist, whereas the blue circle indicates the co-agonist. (B) The Aβ protein binds to GluN2B-containing NMDARs to induce a flux-independent signaling pathway that leads to AMPAR removal from the PM and dendritic spine shrinkage via the p38 MAPK signaling pathway. ↓: decrease; ↑: increase.