Retour aux sources: defining the structural basis of glutamate receptor activation.

Ionotropic glutamate receptors (iGluRs) are the major excitatory neurotransmitter receptor in the vertebrate CNS and, as a result, their activation properties lie at the heart of much of the neuronal network activity observed in the developing and adult brain. iGluRs have also been implicated in many nervous system disorders associated with postnatal development (e.g. autism, schizophrenia), cerebral insult (e.g. stroke, epilepsy), and disorders of the ageing brain (e.g. Alzheimer's disease, Parkinsonism). In view of this, an emphasis has been placed on understanding how iGluRs activate and desensitize in functional and structural terms. Early structural models of iGluRs suggested that the strength of the agonist response was primarily governed by the degree of closure induced in the ligand-binding domain (LBD). However, recent studies have suggested a more nuanced role for the LBD with current evidence identifying the iGluR LBD interface as a "hotspot" regulating agonist behaviour. Such ideas remain to be consolidated with recently solved structures of full-length iGluRs to account for the global changes that underlie channel activation and desensitization.

Mitochondrial reactive oxygen species regulate the strength of inhibitory GABA-mediated synaptic transmission.

Neuronal communication imposes a heavy metabolic burden in maintaining ionic gradients essential for action potential firing and synaptic signalling. Although cellular metabolism is known to regulate excitatory neurotransmission, it is still unclear whether the brain's energy supply affects inhibitory signalling. Here we show that mitochondrial-derived reactive oxygen species (mROS) regulate the strength of postsynaptic GABA(A) receptors at inhibitory synapses of cerebellar stellate cells. Inhibition is strengthened through a mechanism that selectively recruits α3-containing GABA(A) receptors into synapses with no discernible effect on resident α1-containing receptors. Since mROS promotes the emergence of postsynaptic events with unique kinetic properties, we conclude that newly recruited α3-containing GABA(A) receptors are activated by neurotransmitter released onto discrete postsynaptic sites. Although traditionally associated with oxidative stress in neurodegenerative disease, our data identify mROS as a putative homeostatic signalling molecule coupling cellular metabolism to the strength of inhibitory transmission.

Defining the structural relationship between kainate-receptor deactivation and desensitization.

Desensitization is an important mechanism curtailing the activity of ligand-gated ion channels (LGICs). Although the structural basis of desensitization is not fully resolved, it is thought to be governed by physicochemical properties of bound ligands. Here, we show the importance of an allosteric cation-binding pocket in controlling transitions between activated and desensitized states of rat kainate-type (KAR) ionotropic glutamate receptors (iGluRs). Tethering a positive charge to this pocket sustains KAR activation, preventing desensitization, whereas mutations that disrupt cation binding eliminate channel gating. These different outcomes explain the structural distinction between deactivation and desensitization. Deactivation occurs when the ligand unbinds before the cation, whereas desensitization proceeds if a ligand is bound without cation pocket occupancy. This sequence of events is absent from AMPA-type iGluRs; thus, cations are identified as gatekeepers of KAR gating, a role unique among even closely related LGICs.

Crosslinking the ligand-binding domain dimer interface locks kainate receptors out of the main open state.

Kainate-selective ionotropic glutamate receptors (iGluRs) fulfil key roles in the CNS, making them the subject of detailed structural and functional analyses. Although they are known to gate a channel pore with high and low ion-permeation rates, it is still not clear how switches between these gating modes are achieved at the structural level. Here, we uncover an unexpected role for the ligand-binding domain (LBD) dimer assembly in this process. Covalent crosslinking of the dimer interface keeps kainate receptors out of the main open state but permits access to lower conductance states suggesting that significant rearrangements of the dimer interface are required for the receptor to achieve full activation. These observations differ from NMDA-selective iGluRs where constraining dimer movement reduces open-channel probability. In contrast, our data show that restricting movement of the dimer interface interferes with conformational changes that underlie both activation and desensitization. Working within the limits of a common architectural design, we propose functionally diverse iGluR families were able to emerge during evolution by re-deploying existing gating structures to fulfil different tasks.

Functional evidence for D-serine inhibition of non-N-methyl-D-aspartate ionotropic glutamate receptors in retinal neurons.

D-Serine is an important signaling molecule throughout the central nervous system, acting as an N-methyl-D-aspartate (NMDA) receptor coagonist. This study investigated the D-serine modulation of non-NMDA ionotropic glutamate receptors expressed by inner retinal neurons. We first identified that the degradation of endogenous retinal D-serine, by application of D-amino acid oxidase, caused an enhancement of kainate- and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor-mediated calcium responses from the ganglion cell layer of the isolated rat retina and light-evoked responses obtained by multi-electrode array recordings from the guinea pig retina. Approximately 30-45% of cells were endogenously inhibited by D-serine, as suggested by the effect of D-amino acid oxidase. Conversely, bath application of D-serine caused a reduction in multi-electrode array recorded responses and decreased kainate, but not potassium-induced calcium responses, in a concentration-dependent manner (IC(50), 280 µm). Using cultured retinal ganglion cells to reduce network influences, D-serine reduced kainate-induced calcium responses and AMPA induced whole-cell currents. Finally, the inhibitory effect of D-serine on the kainate-induced calcium response was abolished by IEM 1460, thereby identifying calcium-permeable AMPA receptors as a potential target for D-serine. To our knowledge, this is the first study to address specifically the effect of D-serine on AMPA/kainate receptors in intact central nervous system tissue, to identify its effect on calcium permeable AMPA receptors and to report the endogenous inhibition of AMPA/kainate receptors.

The light-induced reduction of horizontal cell receptive field size in the goldfish retina involves nitric oxide.

Horizontal cells of the vertebrate retina have large receptive fields as a result of extensive gap junction coupling. Increased ambient illumination reduces horizontal cell receptive field size. Using the isolated goldfish retina, we have assessed the contribution of nitric oxide to the light-dependent reduction of horizontal cell receptive field size. Horizontal cell receptive field size was assessed by comparing the responses to centered spot and annulus stimuli and from the responses to translated slit stimuli. A period of steady illumination decreased the receptive field size of horizontal cells, as did treatment with the nitric oxide donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (100 µM). Blocking the endogenous production of nitric oxide with the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (1 mM), decreased the light-induced reduction of horizontal cell receptive field size. These findings suggest that nitric oxide is involved in light-induced reduction of horizontal cell receptive field size.

d-Serine enhancement of NMDA receptor-mediated calcium increases in rat retinal ganglion cells.

NMDA receptor (NMDAR) activation is enhanced by d-serine or glycine acting at a specific binding site. Previous work has shown d-serine enhancement of NMDAR currents in retinal ganglion cells. One of the major functions of most NMDA channels is to permit calcium influx into cells. We show that d-serine enhances glutamate-induced calcium responses in immunopanned retinal ganglion cells. This effect was specific to NMDA receptors as similar results were found with NMDA, but not kainate, and was reduced or blocked by modulators of the NMDAR coagonist binding site. d-Serine and glycine enhanced glutamate-induced calcium responses in a dose-dependent manner and at equimolar concentrations there was no difference in the efficacy of the coagonists. In isolated retinas NMDA-induced calcium responses were enhanced by d-serine coapplication in 46% of ganglion cells. Endogenous d-serine degradation by treatment with d-amino acid oxidase caused a approximately 45% decrease in the NMDA-induced response that could be reversed by coapplication with d-serine. d-Serine and glycine were equally effective in enhancing glutamatergic calcium responses. Endogenous d-serine contributes to NMDAR activation in retinal wholemounts and some but not all retinal ganglion cells may experience saturating levels of d-serine or glycine.

Slow excitation of cultured rat retinal ganglion cells by activating group I metabotropic glutamate receptors.

As in many CNS neurons, retinal ganglion cells (RGCs) receive fast synaptic activation through postsynaptic ionotropic receptors. However, the potential role of postsynaptic group I metabotropic glutamate receptors (mGluRs) in these neurons is unknown. In this study we first demonstrated that the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) increased intracellular calcium concentration in neurons within the ganglion cell layer of the rat retina. This prompted us to use an immunopanned-RGC and cortical astroglia coculture preparation to explore the effect of group I mGluR activation on the electrophysiological properties of cultured RGCs. Using perforated patch-clamp recordings in current-clamp configuration, we found that application of DHPG increased spontaneous spiking and depolarized the resting membrane potential of RGCs. This boosting effect was attributed to an increase in membrane resistance due to blockade of a background K(+) conductance. Further experiments showed that the group I mGluR-sensitive K(+) conductance was not blocked by 3 mM Cs(+), but was sensitive to acidification. Pharmacological studies indicated that the effect of DHPG on RGCs was mediated by the mGluR1 rather than the mGluR5 receptor subtype. Our results suggest a facilitatory role for group I mGluR activation in modulating RGC excitability in the mammalian inner retina.

Proton-mediated feedback inhibition of presynaptic calcium channels at the cone photoreceptor synapse.

Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K(+) increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K(+)-induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.