Transmembrane AMPA receptor regulatory proteins and cornichon-2 allosterically regulate AMPA receptor antagonists and potentiators

AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator [(3)H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [(3)H]-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of [(3)H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [(3)H]-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.     

Dynamics of Cleft Closure of the GluA2 Ligand-binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange

The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.     

Potent and selective inhibition of a single alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit by an RNA aptamer

Inhibitors of AMPA-type glutamate ion channels are useful as biochemical probes for structure-function studies and as drug candidates for a number of neurological disorders and diseases. Here, we describe the identification of an RNA inhibitor or aptamer by an in vitro evolution approach and a characterization of its mechanism of inhibition on the sites of interaction by equilibrium binding and on the receptor channel opening rate by a laser-pulse photolysis technique. Our results show that the aptamer is a noncompetitive inhibitor that selectively inhibits the GluA2Q(flip) AMPA receptor subunit without any effect on other AMPA receptor subunits or kainate or NMDA receptors. On the GluA2 subunit, this aptamer preferentially inhibits the flip variant. Furthermore, the aptamer preferentially inhibits the closed-channel state of GluA2Q(flip) with a K(I) = 1.5 μM or by ～15-fold over the open-channel state. The potency and selectivity of this aptamer rival those of small molecule inhibitors. Together, these properties make this aptamer a promising candidate for the development of water-soluble, highly potent, and GluA2 subunit-selective drugs.     

Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor

The conformational changes in the agonist binding domain of the glycine-binding GluN1 and glutamate-binding GluN2A subunits of the N-methyl D-aspartic acid receptor upon binding agonists of varying efficacy have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists; however, no significant changes in the cleft closure are observed between partial and full agonists. This is consistent with the previously reported crystal structures for the isolated agonist binding domain of this receptor. Additionally, the LRET-based distances show that the agonist binding domain of the glutamate-binding GluN2A subunit exhibits a graded cleft closure with the extent of cleft closure being proportional to the extent of activation, indicating that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.     

Regulation of N-methyl-D-aspartic acid (NMDA) receptors by metabotropic glutamate receptor 7

Emerging evidence suggests that metabotropic glutamate receptors (mGluRs) are potential novel targets for brain disorders associated with the dysfunction of prefrontal cortex (PFC), a region critical for cognitive and emotional processes. Because N-methyl-D-aspartic acid receptor (NMDAR) dysregulation has been strongly associated with the pathophysiology of mental illnesses, we examined the possibility that mGluRs might be involved in modulating PFC functions by targeting postsynaptic NMDARs. We found that application of prototypical group III mGluR agonists significantly reduced NMDAR-mediated synaptic and ionic currents in PFC pyramidal neurons, which was mediated by mGluR7 localized at postsynaptic neurons and involved the β-arrestin/ERK signaling pathway. The mGluR7 modulation of NMDAR currents was prevented by agents perturbing actin dynamics and by the inhibitor of cofilin, a major actin-depolymerizing factor. Consistently, biochemical and immunocytochemical results demonstrated that mGluR7 activation increased cofilin activity and F-actin depolymerization via an ERK-dependent mechanism. Furthermore, mGluR7 reduced the association of NMDARs with the scaffolding protein PSD-95 and the surface level of NMDARs in an actin-dependent manner. These data suggest that mGluR7, by affecting the cofilin/actin signaling, regulates NMDAR trafficking and function. Because ablation of mGluR7 leads to a variety of behavioral symptoms related to PFC dysfunction, such as impaired working memory and reduced anxiety and depression, our results provide a potential mechanism for understanding the role of mGluR7 in mental health and disorders.     

N-methyl-D-aspartate receptor mechanosensitivity is governed by C terminus of NR2B subunit

N-methyl-D-aspartate receptors (NMDARs), critical mediators of both physiologic and pathologic neurological signaling, have previously been shown to be sensitive to mechanical stretch through the loss of its native Mg(2+) block. However, the regulation of this mechanosensitivity has yet to be further explored. Furthermore, as it has become apparent that NMDAR-mediated signaling is dependent on specific NMDAR subtypes, as governed by the identity of the NR2 subunit, a crucial unanswered question is the role of subunit composition in observed NMDAR mechanosensitivity. Here, we used a recombinant system to assess the mechanosensitivity of specific subtypes and demonstrate that the mechanosensitive property is uniquely governed by the NR2B subunit. NR1/NR2B NMDARs displayed significant stretch sensitivity, whereas NR1/NR2A NMDARs did not respond to stretch. Furthermore, NR2B mechanosensitivity was regulated by PKC activity, because PKC inhibition reduced stretch responses in transfected HEK 293 cells and primary cortical neurons. Finally, using NR2B point mutations, we identified a PKC phosphorylation site, Ser-1323 on NR2B, as a unique critical regulator of stretch sensitivity. These data suggest that the selective mechanosensitivity of NR2B can significantly impact neuronal response to traumatic brain injury and illustrate that the mechanical tone of the neuron can be dynamically regulated by PKC activity.     

Chronic opioid potentiates presynaptic but impairs postsynaptic N-methyl-D-aspartic acid receptor activity in spinal cords: implications for opioid hyperalgesia and tolerance

Opioids are the most effective analgesics for the treatment of moderate to severe pain. However, chronic opioid treatment can cause both hyperalgesia and analgesic tolerance, which limit their clinical efficacy. In this study, we determined the role of pre- and postsynaptic NMDA receptors (NMDARs) in controlling increased glutamatergic input in the spinal cord induced by chronic systemic morphine administration. Whole-cell voltage clamp recordings of excitatory postsynaptic currents (EPSCs) were performed on dorsal horn neurons in rat spinal cord slices. Chronic morphine significantly increased the amplitude of monosynaptic EPSCs evoked from the dorsal root and the frequency of spontaneous EPSCs, and these changes were largely attenuated by blocking NMDARs and by inhibiting PKC, but not PKA. Also, blocking NR2A- or NR2B-containing NMDARs significantly reduced the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs in morphine-treated rats. Strikingly, morphine treatment largely decreased the amplitude of evoked NMDAR-EPSCs and NMDAR currents of dorsal horn neurons elicited by puff NMDA application. The reduction in postsynaptic NMDAR currents caused by morphine was prevented by resiniferatoxin pretreatment to ablate TRPV1-expressing primary afferents. Furthermore, intrathecal injection of the NMDAR antagonist significantly attenuated the development of analgesic tolerance and the reduction in nociceptive thresholds induced by chronic morphine. Collectively, our findings indicate that chronic opioid treatment potentiates presynaptic, but impairs postsynaptic, NMDAR activity in the spinal cord. PKC-mediated increases in NMDAR activity at nociceptive primary afferent terminals in the spinal cord contribute critically to the development of opioid hyperalgesia and analgesic tolerance.     

Impaired alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking and function by mutant huntingtin

Emerging evidence from studies of Huntington disease (HD) pathophysiology suggests that huntingtin (htt) and its associated protein HAP1 participate in intracellular trafficking and synaptic function. However, it is largely unknown whether AMPA receptor trafficking, which is crucial for controlling the efficacy of synaptic excitation, is affected by the mutant huntingtin with polyglutamine expansion (polyQ-htt). In this study, we found that expressing polyQ-htt in neuronal cultures significantly decreased the amplitude and frequency of AMPAR-mediated miniature excitatory postsynaptic current (mEPSC), while expressing wild-type huntingtin (WT-htt) increased mEPSC. AMPAR-mediated synaptic transmission was also impaired in a transgenic mouse model of HD expressing polyQ-htt. The effect of polyQ-htt on mEPSC was mimicked by knockdown of HAP1 and occluded by the dominant negative HAP1. Moreover, we found that huntingtin affected mESPC via a mechanism depending on the kinesin motor protein, KIF5, which controls the transport of GluR2-containing AMPARs along microtubules in dendrites. The GluR2/KIF5/HAP1 complex was disrupted and dissociated from microtubules in the HD mouse model. Together, these data suggest that AMPAR trafficking and function is impaired by mutant huntingtin, presumably due to the interference of KIF5-mediated microtubule-based transport of AMPA receptors. The diminished strength of glutamatergic transmission could contribute to the deficits in movement control and cognitive processes in HD conditions.     

The Structure of (?)-Kaitocephalin Bound to the Ligand Binding Domain of the (S)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)/Glutamate Receptor,GluA2

Glutamate receptors mediate the majority of excitatory synaptic transmission in the central nervous system, and excessive stimulation of these receptors is involved in a variety of neurological disorders and neuronal damage from stroke. The development of new subtype-specific antagonists would be of considerable therapeutic interest. Natural products can provide important new lead compounds for drug discovery. The only natural product known to inhibit glutamate receptors competitively is (−)-kaitocephalin, which was isolated from the fungus Eupenicillium shearii and found to protect CNS neurons from excitotoxicity. Previous work has shown that it is a potent antagonist of some subtypes of glutamate receptors (AMPA and NMDA, but not kainate). The structure of kaitocephalin bound to the ligand binding domain of the AMPA receptor subtype, GluA2, is reported here. The structure suggests how kaitocephalin can be used as a scaffold to develop more selective and high affinity antagonists for glutamate receptors.     

Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.     

Casein kinase 2-mediated synaptic GluN2A up-regulation increases N-methyl-D-aspartate receptor activity and excitability of hypothalamic neurons in hypertension

Increased glutamatergic input, particularly N-methyl-D-aspartate receptor (NMDAR) activity, in the paraventricular nucleus (PVN) of the hypothalamus is closely associated with high sympathetic outflow in essential hypertension. The molecular mechanisms underlying augmented NMDAR activity in hypertension are unclear. GluN2 subunit composition at the synaptic site critically determines NMDAR functional properties. Here, we found that evoked NMDAR-excitatory postsynaptic currents (EPSCs) of retrogradely labeled spinally projecting PVN neurons displayed a larger amplitude and shorter decay time in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rats. Blocking GluN2B caused a smaller decrease in NMDAR-EPSCs of PVN neurons in SHRs than in WKY rats. In contrast, GluN2A blockade resulted in a larger reduction in evoked NMDAR-EPSCs and puff NMDA-elicited currents of PVN neurons in SHRs than in WKY rats. Blocking presynaptic GluN2A, but not GluN2B, significantly reduced the frequency of miniature EPSCs and the firing activity of PVN neurons in SHRs. The mRNA and total protein levels of GluN2A and GluN2B in the PVN were greater in SHRs than in WKY rats. Furthermore, the GluN2B Ser(1480) phosphorylation level and the synaptosomal GluN2A protein level in the PVN were significantly higher in SHRs than in WKY rats. Inhibition of protein kinase CK2 normalized the GluN2B Ser(1480) phosphorylation level and the contribution of GluN2A to NMDAR-EPSCs and miniature EPSCs of PVN neurons in SHRs. Collectively, our findings suggest that CK2-mediated GluN2B phosphorylation contributes to increased synaptic GluN2A, which potentiates pre- and postsynaptic NMDAR activity and the excitability of PVN presympathetic neurons in hypertension.     

A Conserved Mechanism for Gating in an Ionotropic Glutamate Receptor

Ionotropic glutamate receptor (iGluR) channels control synaptic activity. The crystallographic structure of GluA2, the prototypical iGluR, reveals a clamshell-like ligand-binding domain (LBD) that closes in the presence of glutamate to open a gate on the pore lining α-helix. How LBD closure leads to gate opening remains unclear. Here, we show that bending the pore helix at a highly conserved alanine residue (Ala-621) below the gate is responsible for channel opening. Substituting Ala-621 with the smaller more flexible glycine resulted in a basally active, nondesensitizing channel with ∼39-fold increase in glutamate potency without affecting surface expression or binding. On GluA2(A621G), the partial agonist kainate showed efficacy similar to a full agonist, and competitive antagonists CNQX and DNQX acted as a partial agonists. Met-629 in GluA2 sits above the gate and is critical in transmitting LBD closure to the gate. Substituting Met-629 with the flexible glycine resulted in reduced channel activity and glutamate potency. The pore regions in potassium channels are structurally similar to iGluRs. Whereas potassium channels typically use glycines as a hinge for gating, iGluRs use the less flexible alanine as a hinge at a similar position to maintain low basal activity allowing for ligand-mediated gating.     

Cytosolic Sulfotransferase 1A3 Is Induced by Dopamine and Protects Neuronal Cells from Dopamine Toxicity: ROLE OF D1 RECEPTOR-N-METHYL-d-ASPARTATE RECEPTOR COUPLING*

Dopamine neurotoxicity is associated with several neurodegenerative diseases, and neurons utilize several mechanisms, including uptake and metabolism, to protect them from injury. Metabolism of dopamine involves three enzymes: monoamine oxidase, catechol O-methyltransferase, and sulfotransferase. In primates but not lower order animals, a sulfotransferase (SULT1A3) is present that can rapidly metabolize dopamine to dopamine sulfate. Here, we show that SULT1A3 and a closely related protein SULT1A1 are highly inducible by dopamine. This involves activation of the D1 and NMDA receptors. Both ERK1/2 phosphorylation and calcineurin activation are required for induction. Pharmacological agents that inhibited induction or siRNA targeting SULT1A3 significantly increased the susceptibility of cells to dopamine toxicity. Taken together, these results show that dopamine can induce its own metabolism and protect neuron-like cells from damage, suggesting that SULT1A3 activity may be a risk factor for dopamine-dependent neurodegenerative diseases.     

Dual-specific phosphatase-6 (Dusp6) and ERK mediate AMPA receptor-induced oligodendrocyte death

Oligodendrocytes, the myelinating cells of the CNS, are highly vulnerable to glutamate excitotoxicity, a mechanism involved in tissue damage in multiple sclerosis. Thus, understanding oligodendrocyte death at the molecular level is important to develop new therapeutic approaches to treat the disease. Here, using microarray analysis and quantitative PCR, we observed that dual-specific phosphatase-6 (Dusp6), an extracellular regulated kinase-specific phosphatase, is up-regulated in oligodendrocyte cultures as well as in optic nerves after AMPA receptor activation. In turn, Dusp6 is overexpressed in optic nerves from multiple sclerosis patients before the appearance of evident damage in this structure. We further analyzed the role of Dusp6 and ERK signaling in excitotoxic oligodendrocyte death and observed that AMPA receptor activation induces a rapid increase in ERK1/2 phosphorylation. Blocking Dusp6 expression, which enhances ERK1/2 phosphorylation, significantly diminished AMPA receptor-induced oligodendrocyte death. In contrast, MAPK/ERK pathway inhibition with UO126 significantly potentiates excitotoxic oligodendrocyte death and increases cytochrome c release, mitochondrial depolarization, and mitochondrial calcium overload produced by AMPA receptor stimulation. Upstream analysis demonstrated that MAPK/ERK signaling alters AMPA receptor properties. Indeed, Dusp6 overexpression as well as incubation with UO126 produced an increase in AMPA receptor-induced inward currents and cytosolic calcium overload. Together, these data suggest that levels of phosphorylated ERK, controlled by Dusp6 phosphatase, regulate glutamate receptor permeability and oligodendroglial excitotoxicity. Therefore, targeting Dusp6 may be a useful strategy to prevent oligodendrocyte death in multiple sclerosis and other diseases involving CNS white matter.     

Regulation of AMPA Receptor Trafficking and Function by Glycogen Synthase Kinase 3

Accumulating evidence suggests that glycogen synthase kinase 3 (GSK-3) is a multifunctional kinase implicated in neuronal development, mood stabilization, and neurodegeneration. However, the synaptic actions of GSK-3 are largely unknown. In this study, we examined the impact of GSK-3 on AMPA receptor (AMPAR) channels, the major mediator of excitatory transmission, in cortical neurons. Application of GSK-3 inhibitors or knockdown of GSK-3 caused a significant reduction of the amplitude of miniature excitatory postsynaptic current (mEPSC), a readout of the unitary strength of synaptic AMPARs. Treatment with GSK-3 inhibitors also decreased surface and synaptic GluR1 clusters on dendrites and increased internalized GluR1 in cortical cultures. Rab5, the small GTPase controlling the transport from plasma membrane to early endosomes, was activated by GSK-3 inhibitors. Knockdown of Rab5 prevented GSK-3 inhibitors from regulating mEPSC amplitude. Guanyl nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab5 between membrane and cytosol, formed an increased complex with Rab5 after treatment with GSK-3 inhibitors. Blocking the function of GDI occluded the effect of GSK-3 inhibitors on mEPSC amplitude. In cells transfected with the non-phosphorylatable GDI mutant, GDI(S45A), GSK-3 inhibitors lost the capability to regulate GDI-Rab5 complex, mEPSC amplitude, and AMPAR surface expression. These results suggest that GSK-3, via altering the GDI-Rab5 complex, regulates Rab5-mediated endocytosis of AMPARs. It provides a potential mechanism underlying the role of GSK-3 in synaptic transmission and plasticity.     

Interaction of Endogenous Tau Protein with Synaptic Proteins Is Regulated by N-Methyl-D-aspartate Receptor-dependent Tau Phosphorylation

Amyloid-β and tau protein are the two most prominent factors in the pathology of Alzheimer disease. Recent studies indicate that phosphorylated tau might affect synaptic function. We now show that endogenous tau is found at postsynaptic sites where it interacts with the PSD95-NMDA receptor complex. NMDA receptor activation leads to a selective phosphorylation of specific sites in tau, regulating the interaction of tau with Fyn and the PSD95-NMDA receptor complex. Based on our results, we propose that the physiologically occurring phosphorylation of tau could serve as a regulatory mechanism to prevent NMDA receptor overexcitation.     

The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal β-cell lines and human islet β-cells but not in α-cells. In human islets, glutamate-induced β-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of β-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced β-cell death, whereas GLT1 up-regulation promoted β-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that β-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.     

Sequential Delivery of Synaptic GluA1- and GluA4-containing AMPA Receptors (AMPARs) by SAP97 Anchored Protein Complexes in Classical Conditioning

Multiple signaling pathways are involved in AMPAR trafficking to synapses during synaptic plasticity and learning. The mechanisms for how these pathways are coordinated in parallel but maintain their functional specificity involves subcellular compartmentalization of kinase function by scaffolding proteins, but how this is accomplished is not well understood. Here, we focused on characterizing the molecular machinery that functions in the sequential synaptic delivery of GluA1- and GluA4-containing AMPARs using an in vitro model of eyeblink classical conditioning. We show that conditioning induces the interaction of selective protein complexes with the key structural protein SAP97, which tightly regulates the synaptic delivery of GluA1 and GluA4 AMPAR subunits. The results demonstrate that in the early stages of conditioning the initial activation of PKA stimulates the formation of a SAP97-AKAP/PKA-GluA1 protein complex leading to synaptic delivery of GluA1-containing AMPARs through a SAP97-PSD95 interaction. This is followed shortly thereafter by generation of a SAP97-KSR1/PKC-GluA4 complex for GluA4 AMPAR subunit delivery again through a SAP97-PSD95 interaction. These data suggest that SAP97 forms the molecular backbone of a protein scaffold critical for delivery of AMPARs to the PSD during conditioning. Together, the findings reveal a cooperative interaction of multiple scaffolding proteins for appropriately timed delivery of subunit-specific AMPARs to synapses and support a sequential two-stage model of AMPAR synaptic delivery during classical conditioning.     

Homeostatic synaptic scaling is regulated by protein SUMOylation

Homeostatic scaling allows neurons to alter synaptic transmission to compensate for changes in network activity. Here, we show that suppression of network activity with tetrodotoxin, which increases surface expression of AMPA receptors (AMPARs), dramatically reduces levels of the deSUMOylating (where SUMO is small ubiquitin-like modifier) enzyme SENP1, leading to a consequent increase in protein SUMOylation. Overexpression of the catalytic domain of SENP1 prevents this scaling effect, and we identify Arc as a SUMO substrate involved in the tetrodotoxin-induced increase in AMPAR surface expression. Thus, protein SUMOylation plays an important and previously unsuspected role in synaptic trafficking of AMPARs that underlies homeostatic scaling.     

Interaction of the M4 segment with other transmembrane segments is required for surface expression of mammalian α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors

Ionotropic glutamate receptors (GluRs) are ligand-gated ion channels with a modular structure. The ion channel itself shares structural similarity, albeit an inverted membrane topology, with P-loop channels. Like P-loop channels, prokaryotic GluR subunits (e.g. GluR0) have two transmembrane segments. In contrast, eukaryotic GluRs have an additional transmembrane segment (M4), located C-terminal to the ion channel core. However, the structural/functional significance of this additional transmembrane segment is poorly defined. Although topologically similar to GluR0, mammalian AMPA receptor (GluA1) subunits lacking the M4 segment do not display surface expression. This lack of expression is not due to the M4 segment serving as an anchor to the ligand-binding domain because insertion of an artificial polyleucine transmembrane segment does not rescue surface expression. Specific interactions between M4 and the ligand-binding domain are also unlikely because insertion of polyglycines into the linker connecting them has no deleterious effects on function or surface expression. However, tryptophan and cysteine scanning mutagenesis of the M4 segment, as well as recovery of function in the polyleucine background, defined a unique face of the M4 helix that is required for GluR surface expression. In the AMPA receptor structure, this face forms intersubunit contacts with the transmembrane helices of the ion channel core (M1 and M3) from another subunit within the homotetramer. Thus, our experiments show that a highly specific interaction of the M4 segment with an adjacent subunit is required for surface expression of AMPA receptors. This interaction may represent a mechanism for regulating AMPA receptor biogenesis.