Stargazin modulates AMPA receptor gating and trafficking by distinct domains

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors mediate fast excitatory synaptic transmission in the brain. These ion channels rapidly deactivate and desensitize, which determine the time course of synaptic transmission. Here, we find that the AMPA receptor interacting protein, stargazin, not only mediates AMPA receptor trafficking but also shapes synaptic responses by slowing channel deactivation and desensitization. The cytoplasmic tail of stargazin determines receptor trafficking, whereas the ectodomain controls channel properties. Stargazin alters AMPA receptor kinetics by increasing the rate of channel opening. Disrupting the interaction of stargazin ectodomain with hippocampal AMPA receptors alters the amplitude and shape of synaptic responses, establishing a crucial function for stargazin in controlling the efficacy of synaptic transmission in the brain.     

Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2

Synaptic transmission from excitatory nerve cells in the mammalian brain is largely mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors located at the surface of dendritic spines. The abundance of postsynaptic AMPA receptors correlates with the size of the synapse and the dimensions of the dendritic spine head. Moreover, long-term potentiation is associated with the formation of dendritic spines as well as synaptic delivery of AMPA receptors. The molecular mechanisms that coordinate AMPA receptor delivery and spine morphogenesis are unknown. Here we show that overexpression of the glutamate receptor 2 (GluR2) subunit of AMPA receptors increases spine size and density in hippocampal neurons, and more remarkably, induces spine formation in GABA-releasing interneurons that normally lack spines. The extracellular N-terminal domain (NTD) of GluR2 is responsible for this effect, and heterologous fusion proteins of the NTD of GluR2 inhibit spine morphogenesis. We propose that the NTD of GluR2 functions at the cell surface as part of a receptor-ligand interaction that is important for spine growth and/or stability.     

Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites

In cells, molecular motors operate in polarized sorting of molecules, although the steering mechanisms of motors remain elusive. In neurons, the kinesin motor conducts vesicular transport such as the transport of synaptic vesicle components to axons and of neurotransmitter receptors to dendrites, indicating that vesicles may have to drive the motor for the direction to be correct. Here we show that an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor subunit--GluR2-interacting protein (GRIP1)--can directly interact and steer kinesin heavy chains to dendrites as a motor for AMPA receptors. As would be expected if this complex is functional, both gene targeting and dominant negative experiments of heavy chains of mouse kinesin showed abnormal localization of GRIP1. Moreover, expression of the kinesin-binding domain of GRIP1 resulted in accumulation of the endogenous kinesin predominantly in the somatodendritic area. This pattern was different from that generated by the overexpression of the kinesin-binding scaffold protein JSAP1 (JNK/SAPK-associated protein-1, also known as Mapk8ip3), which occurred predominantly in the somatoaxon area. These results indicate that directly binding proteins can determine the traffic direction of a motor protein.     

Developmental and activity-dependent regulation of kainate receptors at thalamocortical synapses.

Most of the fast excitatory synaptic transmission in the mammalian brain is mediated by ionotrophic glutamate receptors, of which there are three subtypes: AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate), NMDA (N-methyl-D-aspartate) and kainate. Although kainate-receptor subunits (GluR5-7, KA1 and 2) are widely expressed in the mammalian central nervous system, little is known about their function. The development of pharmacological agents that distinguish between AMPA and kainate receptors has now allowed the functions of kainate receptors to be investigated. The modulation of synaptic transmission by kainate receptors and their synaptic activation in a variety of brain regions have been reported. The expression of kainate receptor subunits is developmentally regulated but their role in plasticity and development is unknown. Here we show that developing thalamocortical synapses express postsynaptic kainate receptors as well as AMPA receptors; however, the two receptor subtypes do not colocalize. During the critical period for experience-dependent plasticity, the kainate-receptor contribution to transmission decreases; a similar decrease occurs when long-term potentiation is induced in vitro. This indicates that during development there is activity-dependent regulation of the expression of kainate receptors at thalamocortical synapses.     

Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex.

The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L-glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein.     

Structure and different conformational states of native AMPA receptor complexes

Ionotropic glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Their modulation is believed to affect learning and memory, and their dysfunction has been implicated in the pathogenesis of neurological and psychiatric diseases. Despite a wealth of functional data, little is known about the intact, three-dimensional structure of these ligand-gated ion channels. Here, we present the structure of native AMPA receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AMPA-Rs) purified from rat brain, as determined by single-particle electron microscopy. Unlike the homotetrameric recombinant GluR2 (ref. 3), the native heterotetrameric AMPA-R adopted various conformations, which reflect primarily a variable separation of the two dimeric extracellular amino-terminal domains. Members of the stargazin/TARP family of transmembrane proteins co-purified with AMPA-Rs and contributed to the density representing the transmembrane region of the complex. Glutamate and cyclothiazide markedly altered the conformational equilibrium of the channel complex, suggesting that desensitization is related to separation of the N-terminal domains. These data provide a glimpse of the conformational changes of an important ligand-gated ion channel of the brain.     

Regulation of AMPA receptor lateral movements

An essential feature in the modulation of the efficacy of synaptic transmission is rapid changes in the number of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors at post-synaptic sites on neurons. Regulation of receptor endo- and exocytosis has been shown to be involved in this process. Whether regulated lateral diffusion of receptors in the plasma membrane also participates in receptor exchange to and from post-synaptic sites remains unknown. We analysed the lateral mobility of native AMPA receptors containing the glutamate receptor subunit GluR2 in rat cultured hippocampal neurons, using single-particle tracking and video microscopy. Here we show that AMPA receptors alternate within seconds between rapid diffusive and stationary behaviour. During maturation of neurons, stationary periods increase in frequency and length, often in spatial correlation with synaptic sites. Raising intracellular calcium, a central element in synaptic plasticity, triggers rapid receptor immobilization and local accumulation on the neuronal surface. We suggest that calcium influx prevents AMPA receptors from diffusing, and that lateral receptor diffusion to and from synaptic sites acts in the rapid and controlled regulation of receptor numbers at synapses.     

A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor.

A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains. The phosphoprotein EBP50 (for ezrinradixin-moesin(ERM)-binding phosphoprotein-50) binds to the cytoplasmic tail of the beta2-adrenergic receptor through a PDZ domain and to the cortical actin cytoskeleton through an ERM-binding domain. Disrupting the interaction of EBP50 with either domain or depolymerization of the actin cytoskeleton itself causes missorting of endocytosed beta2-adrenergic receptors but does not affect the recycling of transferrin receptors. A serine residue at position 411 in the tail of the beta2-adrenergic receptor is a substrate for phosphorylation by GRK-5 (for G-protein-coupled-receptor kinase-5) and is required for interaction with EBP50 and for proper recycling of the receptor. Our results identify a new role for PDZ-domain-mediated protein interactions and for the actin cytoskeleton in endocytic sorting, and suggest a mechanism by which GRK-mediated phosphorylation could regulate membrane trafficking of G-protein-coupled receptors after endocytosis.     

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.     

Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors are members of a gene family of ligand-gated transmitter receptors that includes muscle nicotinic receptors, GABAA receptors and glycine receptors. Several lines of evidence indicate that neuronal nicotinic receptors can be made up of only two subunits, an alpha (alpha) subunit which binds ligand, and a non-alpha (n alpha) or beta (beta) subunit. The stoichiometry of each subunit in the functional receptor has been difficult to assess, however. Estimates of the molecular weight of neuronal nicotonic receptor macromolecules suggest that these receptors contain at least four subunits but probably not more than five. We have examined the subunit stoichiometry of the chick neuronal alpha 4/n alpha 1 receptor by first using site-directed mutagenesis to create subunits that confer different single channel properties on the receptor. Co-injection with wild-type and mutant subunits led to the appearance of receptors with wild-type, mutant and hybrid conductances. From the number of hybrid conductances, we could deduce the number of each subunit in the functional receptor.     

Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus

Fast excitatory neurotransmission in the central nervous system occurs at specialized synaptic junctions between neurons, where a high concentration of glutamate directly activates receptor channels. Low-affinity AMPA (alpha-amino-3-hydroxy-5-methyl isoxazole propionic acid) and kainate glutamate receptors are also expressed by some glial cells, including oligodendrocyte precursor cells (OPCs). However, the conditions that result in activation of glutamate receptors on these non-neuronal cells are not known. Here we report that stimulation of excitatory axons in the hippocampus elicits inward currents in OPCs that are mediated by AMPA receptors. The quantal nature of these responses and their rapid kinetics indicate that they are produced by the exocytosis of vesicles filled with glutamate directly opposite these receptors. Some of these AMPA receptors are permeable to calcium ions, providing a link between axonal activity and internal calcium levels in OPCs. Electron microscopic analysis revealed that vesicle-filled axon terminals make synaptic junctions with the processes of OPCs in both the young and adult hippocampus. These results demonstrate the existence of a rapid signalling pathway from pyramidal neurons to OPCs in the mammalian hippocampus that is mediated by excitatory, glutamatergic synapses.     

Long-term potentiation of NMDA receptor-mediated synaptic transmission in the hippocampus.

Neurotransmission at most excitatory synapses in the brain operates through two types of glutamate receptor termed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors; these mediate the fast and slow components of excitatory postsynaptic potentials respectively. Activation of NMDA receptors can also lead to a long-lasting modification in synaptic efficiency at glutamatergic synapses; this is exemplified in the CA1 region of the hippocampus, where NMDA receptors mediate the induction of long-term potentiation (LTP). It is believed that in this region LTP is maintained by a specific increase in the AMPA receptor-mediated component of synaptic transmission. We now report, however, that a pharmacologically isolated NMDA receptor-mediated synaptic response can undergo robust, synapse-specific LTP. This finding has implications for neuropathologies such as epilepsy and neurodegeneration, in which excessive NMDA receptor activation has been implicated. It adds fundamentally to theories of synaptic plasticity because NMDA receptor activation may, in addition to causing increased synaptic efficiency, directly alter the plasticity of synapses.     

Cerebellar GABAA receptor selective for a behavioural alcohol antagonist

Benzodiazepines are widely prescribed anxiolytics and anticonvulsants which bind with high affinity to sites on the GABAA receptor/Cl- channel complex and potentiate the effect of the neurotransmitter GABA (gamma-aminobutyric acid). The heterogeneity of benzodiazepine recognition sites in the central nervous system was revealed by studies showing different classes of GABAA receptor subunits (classes alpha, beta and gamma) and variant subunits in these classes, particularly in the alpha-class. Expression of recombinant subunits produces functional receptors; when certain alpha-variants are coexpressed with beta- and gamma-subunits the resulting receptors have pharmacological properties characteristic of GABAA-benzodiazepine type I or type II receptors. The alpha-variants are differentially expressed in the central nervous system and can be photoaffinity-labelled with benzodiazepines. Here we report a novel alpha-subunit (alpha 6) of cerebellar granule cells. We show that recombinant receptors composed of alpha 6, beta 2 and gamma 2 subunits bind with high affinity to the GABA agonist [3H]muscimol and the benzodiazepine [3H]Ro15-4513 but not the other benzodiazepines or beta-carboniles. The same distinctive pharmacology is observed with GABAA receptors from rat cerebellum immunoprecipitated by an antiserum specific for the alpha 6 subunit. We conclude that this alpha-subunit is part of a cerebellar receptor subtype, selective for Ro15-4513, an antagonist of alcohol-induced motor incoordination and ataxia.     

Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance

Many neurons receive a continuous, or 'tonic', synaptic input, which increases their membrane conductance, and so modifies the spatial and temporal integration of excitatory signals. In cerebellar granule cells, although the frequency of inhibitory synaptic currents is relatively low, the spillover of synaptically released GABA (gamma-aminobutyric acid) gives rise to a persistent conductance mediated by the GABA A receptor that also modifies the excitability of granule cells. Here we show that this tonic conductance is absent in granule cells that lack the alpha6 and delta-subunits of the GABAA receptor. The response of these granule cells to excitatory synaptic input remains unaltered, owing to an increase in a 'leak' conductance, which is present at rest, with properties characteristic of the two-pore-domain K+ channel TASK-1 (refs 9,10,11,12). Our results highlight the importance of tonic inhibition mediated by GABAA receptors, loss of which triggers a form of homeostatic plasticity leading to a change in the magnitude of a voltage-independent K + conductance that maintains normal neuronal behaviour.     

Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving

Relapse to cocaine use after prolonged abstinence is an important clinical problem. This relapse is often induced by exposure to cues associated with cocaine use. To account for the persistent propensity for relapse, it has been suggested that cue-induced cocaine craving increases over the first several weeks of abstinence and remains high for extended periods. We and others identified an analogous phenomenon in rats that was termed 'incubation of cocaine craving': time-dependent increases in cue-induced cocaine-seeking over the first months after withdrawal from self-administered cocaine. Cocaine-seeking requires the activation of glutamate projections that excite receptors for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the nucleus accumbens. Here we show that the number of synaptic AMPA receptors in the accumbens is increased after prolonged withdrawal from cocaine self-administration by the addition of new AMPA receptors lacking glutamate receptor 2 (GluR2). Furthermore, we show that these new receptors mediate the incubation of cocaine craving. Our results indicate that GluR2-lacking AMPA receptors could be a new target for drug development for the treatment of cocaine addiction. We propose that after prolonged withdrawal from cocaine, increased numbers of synaptic AMPA receptors combined with the higher conductance of GluR2-lacking AMPA receptors causes increased reactivity of accumbens neurons to cocaine-related cues, leading to an intensification of drug craving and relapse.     

Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA.

Fast excitatory transmission in the vertebrate central nervous system is mediated mainly by L-glutamate. On the basis of pharmacological, physiological and agonist binding properties, the ionotropic glutamate receptors are classified into NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) and kainate subtypes. Sequence homology between complementary DNA clones encoding non-NMDA glutamate receptor subunits reveals at least two subunit classes: the GluR1 to GluR4 class and the GluR5 class. Here we report the cloning and expression of a functional rat glutamate receptor subunit cDNA, GluR6, which has a very different pharmacology from that of the GluR1-GluR4 class. Receptors generated from the GluR1-GluR4 class have a higher apparent affinity for AMPA than for kainate. When expressed in Xenopus oocytes the homomeric GluR6 receptor is activated by kainate, quisqualate and L-glutamate but not by AMPA, and the apparent affinity for kainate is higher than for receptors from the GluR1-GluR4 class. Desensitization of the receptor was observed with continuous application of agonist. The homomeric GluR6 glutamate receptor exhibits an outwardly rectifying current-voltage relationship. In situ hybridizations reveal a pattern of GluR6 gene expression reminiscent of the binding pattern obtained with [3H]kainate.     

Kainate receptors are involved in synaptic plasticity

The ability of synapses to modify their synaptic strength in response to activity is a fundamental property of the nervous system and may be an essential component of learning and memory. There are three classes of ionotropic glutamate receptor, namely NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid) and kainate receptors; critical roles in synaptic plasticity have been identified for two of these. Thus, at many synapses in the brain, transient activation of NMDA receptors leads to a persistent modification in the strength of synaptic transmission mediated by AMPA receptors. Here, to determine whether kainate receptors are involved in synaptic plasticity, we have used a new antagonist, LY382884 ((3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydro isoquinoline-3-carboxylic acid), which antagonizes kainate receptors at concentrations that do not affect AMPA or NMDA receptors. We find that LY382884 is a selective antagonist at neuronal kainate receptors containing the GluR5 subunit. It has no effect on long-term potentiation (LTP) that is dependent on NMDA receptors but prevents the induction of mossy fibre LTP, which is independent of NMDA receptors. Thus, kainate receptors can act as the induction trigger for long-term changes in synaptic transmission.     

Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. Alpha-neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that alpha-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.     

Direct protein-protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors

GABA(A) (gamma-aminobutyric-acid A) and dopamine D1 and D5 receptors represent two structurally and functionally divergent families of neurotransmitter receptors. The former comprises a class of multi-subunit ligand-gated channels mediating fast interneuronal synaptic transmission, whereas the latter belongs to the seven-transmembrane-domain single-polypeptide receptor superfamily that exerts its biological effects, including the modulation of GABA(A) receptor function, through the activation of second-messenger signalling cascades by G proteins. Here we show that GABA(A)-ligand-gated channels complex selectively with D5 receptors through the direct binding of the D5 carboxy-terminal domain with the second intracellular loop of the GABA(A) gamma2(short) receptor subunit. This physical association enables mutually inhibitory functional interactions between these receptor systems. The data highlight a previously unknown signal transduction mechanism whereby subtype-selective G-protein-coupled receptors dynamically regulate synaptic strength independently of classically defined second-messenger systems, and provide a heuristic framework in which to view these receptor systems in the maintenance of psychomotor disease states.     

NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury

Injury to oligodendrocyte processes, the structures responsible for myelination, is implicated in many forms of brain disorder. Here we show NMDA (N-methyl-D-aspartate) receptor subunit expression on oligodendrocyte processes, and the presence of NMDA receptor subunit messenger RNA in isolated white matter. NR1, NR2A, NR2B, NR2C, NR2D and NR3A subunits showed clustered expression in cell processes, but NR3B was absent. During modelled ischaemia, NMDA receptor activation resulted in rapid Ca2+-dependent detachment and disintegration of oligodendroglial processes in the white matter of mice expressing green fluorescent protein (GFP) specifically in oligodendrocytes (CNP-GFP mice). This effect occurred at mouse ages corresponding to both the initiation and the conclusion of myelination. NR1 subunits were found mainly in oligodendrocyte processes, whereas AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor subunits were mainly found in the somata. Consistent with this observation, injury to the somata was prevented by blocking AMPA/kainate receptors, and preventing injury to oligodendroglial processes required the blocking of NMDA receptors. The presence of NMDA receptors in oligodendrocyte processes explains why previous studies that have focused on the somata have not detected a role for NMDA receptors in oligodendrocyte injury. These NMDA receptors bestow a high sensitivity to acute injury and represent an important new target for drug development in a variety of brain disorders.