Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach (original) (raw)
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Genetic analysis of neuronal ionotropic glutamate receptor subunits
The Journal of …, 2011
In the brain, fast, excitatory synaptic transmission occurs primarily through AMPA-and NMDA-type ionotropic glutamate receptors. These receptors are composed of subunit proteins that determine their biophysical properties and trafficking behaviour. Therefore, determining the function of these subunits and receptor subunit composition is essential for understanding the physiological properties of synaptic transmission. Here, we discuss and evaluate various genetic approaches that have been used to study AMPA and NMDA receptor subunits. These approaches have demonstrated that the GluA1 AMPA receptor subunit is required for activity-dependent trafficking and contributes to basal synaptic transmission, while the GluA2 subunit regulates Ca 2+ permeability, homeostasis and trafficking to the synapse under basal conditions. In contrast, the GluN2A and GluN2B NMDA receptor subunits regulate synaptic AMPA receptor content, both during synaptic development and plasticity. Ongoing research in this field is focusing on the molecular interactions and mechanisms that control these functions. To accomplish this, molecular replacement techniques are being used, where native subunits are replaced with receptors containing targeted mutations. In this review, we discuss a single-cell molecular replacement approach which should arguably advance our physiological understanding of ionotropic glutamate receptor subunits, but is generally applicable to study of any neuronal protein.
Journal of …, 2011
Deletion of N-methyl-D-aspartate receptors (NMDARs) early in development results in an increase in the number of synaptic AMPA receptors (AMPARs), suggesting a role for NMDARs in negatively regulating AMPAR trafficking at developing synapses. Substantial evidence has shown that AMPAR subunits function differentially in AMPAR trafficking. However, the role of AMPAR subunits in the enhancement of AMPARs following NMDAR ablation remains unknown. We have now performed single-cell genetic deletions in double-floxed mice in which the deletion of GluN1 is combined with the deletion of GluA1 or GluA2. We find that the AMPAR enhancement following NMDAR deletion requires the GluA2 subunit, but not the GluA1 subunit, indicating a key role for GluA2 in the regulation of AMPAR trafficking in developing synapses.
The Journal of Neuroscience, 2003
The number and type of receptors present at the postsynaptic membrane determine the response to the neurotransmitter released from the presynaptic terminal. Because most neurons receive multiple and distinct synaptic inputs and contain several different subtypes of receptors stimulated by the same neurotransmitter, the assembly and trafficking of receptors in neurons is a complex process involving many levels of regulation. To investigate the mechanism that neurons use to regulate the assembly of receptor subunits, we studied a GluR2 knockout mouse. GluR2 is a critical subunit that controls calcium permeability of AMPA receptors and is present in most native AMPA receptors. Our data indicate that in the absence of GluR2, aberrant receptor complexes composed of GluR1 and GluR3 are formed in the hippocampus, and that there is an increased number of homomeric GluR1 and GluR3 receptors. We also show that these homomeric and heteromeric receptors are less efficiently expressed at the synapse. Our results show that GluR2 plays a critical role in controlling the assembly of AMPA receptors, and that the assembly of subunits may reflect the affinity of one subunit for another or the stability of intermediates in the assembly process. Therefore, GluR1 may have a greater preference for GluR2 than it does for GluR3.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000
AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have developed antibodies that recognize all AMPA or all NMDA receptor variants on the surface of living neurons. AMPA receptor variants were identified with a polyclonal antibody recognizing the conserved extracellular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibody that binds to an extracellular N-terminal epitope of the NR1 subunit, common to all splice variants. In non-fixed brain sections these antibodies gave labeling patterns similar to autoradiographic distributions with particularly high levels in the hippocampus. Using these antibodies, in conjunction with GluR2-specific and synaptophysin antibodies, we have directly localized and quantified surface-expressed native AMPA and NMDA receptors on cultured living hippocampal neurons during development. Using a quantitative cell ELISA, a dramatic i...
GluA2-lacking AMPA receptors in hippocampal CA1 cell synapses: evidence from gene-targeted mice
Frontiers in molecular neuroscience, 2012
The GluA2 subunit in heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor channels restricts Ca(2+) permeability and block by polyamines, rendering linear the current-voltage relationship of these glutamate-gated cation channels. Although GluA2-lacking synaptic AMPA receptors occur in GABA-ergic inhibitory neurons, hippocampal CA1 pyramidal cell synapses are widely held to feature only GluA2 containing AMPA receptors. A controversy has arisen from reports of GluA2-lacking AMPA receptors at hippocampal CA3-to-CA1 cell synapses and a study contesting these findings. Here we sought independent evidence for the presence of GluA2-lacking AMPA receptors in CA1 pyramidal cell synapses by probing the sensitivity of their gated cation channels in wild-type (WT) mice and gene-targeted mouse mutants to philanthotoxin, a specific blocker of GluA2-lacking AMPA receptors. The mutants either lacked GluA2 for maximal philanthotoxin sensitivity, or, for minimal sensit...
Auxiliary subunits assist AMPA-type glutamate receptors
Science, 2006
Glutamate, the major excitatory neurotransmitter in the brain, acts primarily on two types of ionotropic receptors: a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors. Work over the past decade indicates that regulated changes in the number of synaptic AMPA receptors may serve as a mechanism for information storage. Recent studies demonstrate that a family of small transmembrane AMPA receptor regulatory proteins (TARPs) controls both AMPA receptor trafficking and channel gating. TARPs provide the first example of auxiliary subunits of ionotropic receptors. Here we review the pivotal role that TARPs play in the life cycle of AMPA receptors.
Proceedings of the National Academy of Sciences, 2008
Glutamate is the primary excitatory neurotransmitter in the brain, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors mediate most fast synaptic transmission. AMPA receptors are tetrameric assemblies composed from four possible subunits (GluR1-4). In hippocampal pyramidal cells, AMPA receptors are heteromeric receptors containing the GluR2 subunit and either GluR1 or GluR3. It is generally accepted that the trafficking of GluR1/GluR2 receptors to synapses requires activity, whereas GluR2/GluR3 receptors traffic constitutively. It has been suggested that the trafficking is governed by the cytoplasmic C termini of the subunits. Because the basis for this theory relied on the introduction of unnatural, homomeric, calcium-permeable AMPA receptors, we have used the GluR2(-/-) knock out mouse to determine whether the expression of mutated forms of GluR2 can rescue WT synaptic responses. We find that GluR2, lacking its entire C terminus, or a GluR2 chimera containing the C terminus of GluR1, is capable of trafficking to the synapse in the absence of activity. These findings suggest that the GluR2 C terminus is not required for GluR2 synaptic insertion.
Neuron, 2011
During development there is an activity-dependent switch in synaptic NMDA receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of this switch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2Bcontaining receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence for a large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.
Cell Type and Pathway Dependence of Synaptic AMPA Receptor Number and Variability in the Hippocampus
Neuron, 1998
It has been suggested that some glutamatergic synapses lack functional AMPA receptors. We used quantitative immunogold localization to detennine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as synapses made by Schaffer collaterals on CAl pyramidal spines and by commissural! associational (CIA) tenninals on CA3 pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, we estimate that the AMPA receptor content of CIA synapses on CA3 pyramidal spines ranges from < 3 to 140. A similar range is found in adult Schaffer collateral and CIA synapses.
Persistent Synaptic Scaling Independent of AMPA Receptor Subunit Composition
Journal of Neuroscience, 2013
Despite long-standing evidence that the specific intracellular domains of AMPA-type glutamate receptor (AMPAR) subunits are critical for trafficking, it has recently been demonstrated that there is no absolute requirement for any AMPAR subunit for the receptor insertion underlying LTP. It is unclear whether this holds true to other forms of plasticity. Homeostatic synaptic plasticity (HSP) is an important form of negative feedback that provides stability to neuronal networks, and results at least in part from the insertion of AMPARs into glutamatergic synapses following chronic reductions in neuronal activity. Similar to LTP, the GluA1 subunit has been suggested to be the requisite subunit for HSP-induced AMPAR insertion and acute treatment with signaling molecules that underlie some forms of HSP results in the preferential incorporation of GluA2-lacking receptors. However, knockdown experiments have instead implicated a requirement for the GluA2 subunit. Here we re-examined the requirement for specific AMPAR subunit during chronic tetrodotoxininduced HSP using hippocampal cultures derived from AMPAR subunit knock-out mice. We observed HSP in cultures from GluA1 Ϫ/Ϫ , GluA2 Ϫ/Ϫ , and GluA2 Ϫ/Ϫ GluA3 Ϫ/Ϫ mice, and conclude that, as with LTP, there is no subunit requirement for HSP.