Influence of the TARP γ8-Selective Negative Allosteric Modulator JNJ-55511118 on AMPA Receptor Gating and Channel Conductance.

AMPA-type gultamate receptors (AMPARs) mediate excitatory signaling in the brain and are therapeutic targets for the treatment of diverse neurological disorders. The receptors interact with a variety of auxiliary subunits, including the transmembrane AMPAR regulatory proteins (TARPs). The TARPs influence AMPAR biosynthesis and trafficking and enhance receptor responses by slowing desensitization and deactivation and increasing single-channel conductance. TARP γ8 has an expression pattern that is distinct from that of other TARPs, being enriched in the hippocampus. Recently, several compounds have been identified that selectivity inhibit γ8-containing AMPARs. One such inhibitor, JNJ-55511118, has shown considerable promise for the treatment of epilepsy. However, key details of its mechanism of action are still lacking. Here, using patch-clamp electrophysiological recording from heterologously expressed AMPARs, we show that JNJ-55511118 inhibits peak currents of γ8-containing AMPARs by decreasing their single-channel conductance. The drug also modifies hallmark features of AMPAR pharmacology, including the TARP-dependent actions of intracellular polyamines and the partial agonist kainate. Moreover, we find that JNJ-55511118 reduces the influence of γ8 on all biophysical measures, aside from its effect on the recovery from desensitization. The drug is also effective when applied intracellularly, suggesting it may access its binding site from within the membrane. Additionally, we find that AMPARs incorporating TARP γ2 mutated to contain the JNJ-55511118 binding site, exhibit greater block than seen with AMPARs containing γ8, potentially reflecting differences in TARP stoichiometry. Taken together, our data provide new insight into the mechanism by which γ8-selective drugs inhibit AMPARs. SIGNIFICANCE STATEMENT: Although modulation of AMPA-type glutamate receptors shows promise for the treatment various neurological conditions, the absence of subtype-selective drugs has hindered adoption of this therapeutic strategy. We made patch-clamp recordings to characterize the actions of the γ8-selective AMPAR inhibitor JNJ-55511118 on GluA2(Q) receptors expressed in HEK cells. We report that JNJ-55511118 inhibits AMPAR-mediated currents by reducing single-channel conductance, providing clear insight into the mechanism of action of this important class of AMPAR modulators.

A gain-of-function GRIA2 variant associated with neurodevelopmental delay and seizures: Functional characterization and targeted treatment.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) are ligand-gated cationic channels formed from combinations of GluA1-4 subunits. Pathogenic variants of GRIA1-4 have been described in patients with developmental delay, intellectual disability, autism spectrum disorder, and seizures, with GRIA2 variants typically causing AMPAR loss of function. Here, we identify a novel, heterozygous de novo pathogenic missense mutation in GRIA2 (c.1928 C>T, p.A643V, NM_001083619.1) in a 1-year-old boy with epilepsy, developmental delay, and failure to thrive. We made patch-clamp recordings to compare the functional and pharmacological properties of variant and wild-type receptors expressed in HEK293 cells, with and without the transmembrane AMPAR regulatory protein γ2. This showed GluA2 A643V-containing AMPARs to exhibit a novel gain of function, with greatly slowed deactivation, markedly reduced desensitization, and increased glutamate sensitivity. Perampanel, an antiseizure AMPAR negative allosteric modulator, was able to fully block GluA2 A643V/γ2 currents, suggesting potential therapeutic efficacy. The subsequent introduction of perampanel to the patient's treatment regimen was associated with a marked reduction in seizure burden, a resolution of failure to thrive, and clear developmental gains. Our study reveals that GRIA2 disorder can be caused by a gain-of-function variant, and both predicts and suggests the therapeutic efficacy of perampanel. Perampanel may prove beneficial for patients with other gain-of-function GRIA variants.

Ca2+ -permeable AMPA receptors and their auxiliary subunits in synaptic plasticity and disease.

AMPA receptors are tetrameric glutamate-gated ion channels that mediate a majority of fast excitatory neurotransmission in the brain. They exist as calcium-impermeable (CI-) and calcium-permeable (CP-) subtypes, the latter of which lacks the GluA2 subunit. CP-AMPARs display an array of distinctive biophysical and pharmacological properties that allow them to be functionally identified. This has revealed that they play crucial roles in diverse forms of central synaptic plasticity. Here we summarise the functional hallmarks of CP-AMPARs and describe how these are modified by the presence of auxiliary subunits that have emerged as pivotal regulators of AMPARs. A lasting change in the prevalence of GluA2-containing AMPARs, and hence in the fraction of CP-AMPARs, is a feature in many maladaptive forms of synaptic plasticity and neurological disorders. These include modifications of glutamatergic transmission induced by inflammatory pain, fear conditioning, cocaine exposure, and anoxia-induced damage in neurons and glia. Furthermore, defective RNA editing of GluA2 can cause altered expression of CP-AMPARs and is implicated in motor neuron damage (amyotrophic lateral sclerosis) and the proliferation of cells in malignant gliomas. A number of the players involved in CP-AMPAR regulation have been identified, providing useful insight into interventions that may prevent the aberrant CP-AMPAR expression. Furthermore, recent molecular and pharmacological developments, particularly the discovery of TARP subtype-selective drugs, offer the exciting potential to modify some of the harmful effects of increased CP-AMPAR prevalence in a brain region-specific manner.

TARP γ-2 Is Required for Inflammation-Associated AMPA Receptor Plasticity within Lamina II of the Spinal Cord Dorsal Horn.

In the brain, transmembrane AMPAR regulatory proteins (TARPs) critically influence the distribution, gating, and pharmacology of AMPARs, but the contribution of these auxiliary subunits to AMPAR-mediated signaling in the spinal cord remains unclear. We found that the Type I TARP γ-2 (stargazin) is present in lamina II of the superficial dorsal horn, an area involved in nociception. Consistent with the notion that γ-2 is associated with surface AMPARs, CNQX, a partial agonist at AMPARs associated with Type I TARPs, evoked whole-cell currents in lamina II neurons, but such currents were severely attenuated in γ-2-lacking stargazer (stg/stg) mice. Examination of EPSCs revealed the targeting of γ-2 to be synapse-specific; the amplitude of spontaneously occurring miniature EPSCs (mEPSCs) was reduced in neurons from stg/stg mice, but the amplitude of capsaicin-induced mEPSCs from C-fiber synapses was unaltered. This suggests that γ-2 is associated with AMPARs at synapses in lamina II but excluded from those at C-fiber inputs, a view supported by our immunohistochemical colabeling data. Following induction of peripheral inflammation, a model of hyperalgesia, there was a switch in the current-voltage relationships of capsaicin-induced mEPSCs, from linear to inwardly rectifying, indicating an increased prevalence of calcium-permeable (CP) AMPARs. This effect was abolished in stg/stg mice. Our results establish that, although γ-2 is not typically associated with calcium-impermeable AMPARs at C-fiber synapses, it is required for the translocation of CP-AMPARs to these synapses following peripheral inflammation.SIGNIFICANCE STATEMENT In the brain, transmembrane AMPAR regulatory proteins (TARPs) critically determine the functional properties of AMPARs, but the contribution of these auxiliary subunits to AMPAR-mediated signaling in the spinal cord remains unclear. An increase in the excitability of neurons within the superficial dorsal horn (SDH) of the spinal cord is thought to underlie heighted pain sensitivity. One mechanism considered to contribute to such long-lived changes is the remodeling of the ionotropic AMPA-type glutamate receptors that underlie fast excitatory synaptic transmission in the SDH. Here we show that the TARP γ-2 (stargazin) is present in SDH neurons and is necessary in a form of inflammatory pain-induced plasticity, which involves an increase in the prevalence of synaptic calcium-permeable AMPARs.

Transmembrane AMPAR regulatory protein γ-2 is required for the modulation of GABA release by presynaptic AMPARs.

Presynaptic ionotropic glutamate receptors (iGluRs) play important roles in the control of synaptogenesis and neurotransmitter release, yet their regulation is poorly understood. In particular, the contribution of transmembrane auxiliary proteins, which profoundly shape the trafficking and gating of somatodendritic iGluRs, is unknown. Here we examined the influence of transmembrane AMPAR regulatory proteins (TARPs) on presynaptic AMPARs in cerebellar molecular layer interneurons (MLIs). 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a partial agonist at TARP-associated AMPARs, enhanced spontaneous GABA release in wild-type mice but not in stargazer mice that lack the prototypical TARP stargazin (γ-2). These findings were replicated in mechanically dissociated Purkinje cells with functional adherent synaptic boutons, demonstrating the presynaptic locus of modulation. In dissociated Purkinje cells from stargazer mice, AMPA was able to enhance mIPSC frequency, but only in the presence of the positive allosteric modulator cyclothiazide. Thus, ordinarily, presynaptic AMPARs are unable to enhance spontaneous release without γ-2, which is required predominantly for its effects on channel gating. Presynaptic AMPARs are known to reduce action potential-driven GABA release from MLIs. Although a G-protein-dependent non-ionotropic mechanism has been suggested to underlie this inhibition, paradoxically we found that γ-2, and thus AMPAR gating, was required. Following glutamate spillover from climbing fibers or application of CNQX, evoked GABA release was reduced; in stargazer mice such effects were markedly attenuated in acute slices and abolished in the dissociated Purkinje cell-nerve bouton preparation. We suggest that γ-2 association, by increasing charge transfer, allows presynaptic AMPARs to depolarize the bouton membrane sufficiently to modulate both phasic and spontaneous release.

Auxiliary Subunit GSG1L Acts to Suppress Calcium-Permeable AMPA Receptor Function.

AMPA-type glutamate receptors are ligand-gated cation channels responsible for a majority of the fast excitatory synaptic transmission in the brain. Their behavior and calcium permeability depends critically on their subunit composition and the identity of associated auxiliary proteins. Calcium-permeable AMPA receptors (CP-AMPARs) contribute to various forms of synaptic plasticity, and their dysfunction underlies a number of serious neurological conditions. For CP-AMPARs, the prototypical transmembrane AMPAR regulatory protein stargazin, which acts as an auxiliary subunit, enhances receptor function by increasing single-channel conductance, slowing channel gating, increasing calcium permeability, and relieving the voltage-dependent block by endogenous intracellular polyamines. We find that, in contrast, GSG1L, a transmembrane auxiliary protein identified recently as being part of the AMPAR proteome, acts to reduce the weighted mean single-channel conductance and calcium permeability of recombinant CP-AMPARs, while increasing polyamine-dependent rectification. To examine the effects of GSG1L on native AMPARs, we manipulated its expression in cerebellar and hippocampal neurons. Transfection of GSG1L into mouse cultured cerebellar stellate cells that lack this protein increased the inward rectification of mEPSCs. Conversely, shRNA-mediated knockdown of endogenous GSG1L in rat cultured hippocampal pyramidal neurons led to an increase in mEPSC amplitude and in the underlying weighted mean single-channel conductance, revealing that GSG1L acts to suppress current flow through native CP-AMPARs. Thus, our data suggest that GSG1L extends the functional repertoire of AMPAR auxiliary subunits, which can act not only to enhance but also diminish current flow through their associated AMPARs. SIGNIFICANCE STATEMENT: Calcium-permeable AMPA receptors (CP-AMPARs) are an important group of receptors for the neurotransmitter glutamate. These receptors contribute to various forms of synaptic plasticity, and alterations in their expression or regulation are also seen in a number of serious neurological conditions, including stroke, motor neuron disease, and cocaine addiction. Several groups of auxiliary transmembrane proteins have been described that enhance the function and cell-surface expression of AMPARs. We now report that the recently identified auxiliary protein GSG1L decreases weighted mean channel conductance and calcium permeability of CP-AMPARs while increasing polyamine-dependent rectification by diminishing outward current. Our experiments reveal that GSG1L is an auxiliary subunit that can markedly suppress CP-AMPAR function, in both recombinant systems and central neurons.

Synapse-specific expression of calcium-permeable AMPA receptors in neocortical layer 5.

KEY POINTS: In the hippocampus, calcium-permeable AMPA receptors have been found in a restricted subset of neuronal types that inhibit other neurons, although their localization in the neocortex is less well understood. In the present study, we looked for calcium-permeable AMPA receptors in two distinct populations of neocortical inhibitory neurons: basket cells and Martinotti cells. We found them in the former but not in the latter. Furthermore, in basket cells, these receptors were associated with particularly fast responses. Computer modelling predicted (and experiments verified) that fast calcium-permeable AMPA receptors enable basket cells to respond rapidly, such that they promptly inhibit neighbouring cells and shut down activity. The results obtained in the present study help our understanding of pathologies such as stroke and epilepsy that have been associated with disordered regulation of calcium-permeable AMPA receptors. ABSTRACT: AMPA-type glutamate receptors (AMPARs) lacking an edited GluA2 subunit are calcium-permeable (CP) and contribute to synaptic plasticity in several hippocampal interneuron types, although their precise role in the neocortex is not well described. We explored the presence of CP-AMPARs at pyramidal cell (PC) inputs to Martinotti cells (MCs) and basket cells (BCs) in layer 5 of the developing mouse visual cortex (postnatal days 12-21). GluA2 immunolabelling was stronger in MCs than in BCs. A differential presence of CP-AMPARs at PC-BC and PC-MC synapses was confirmed electrophysiologically, based on measures of spermine-dependent rectification and CP-AMPAR blockade by 1-naphtyl acetyl spermine using recordings from synaptically connected cell pairs, NPEC-AMPA uncaging and miniature current recordings. In addition, CP-AMPAR expression in BCs was correlated with rapidly decaying synaptic currents. Computer modelling predicted that this reduces spike latencies and sharpens suprathreshold responses in BCs, which we verified experimentally using the dynamic clamp technique. Thus, the synapse-specific expression of CP-AMPARs may critically influence both plasticity and information processing in neocortical microcircuits.

Molecular mechanisms contributing to TARP regulation of channel conductance and polyamine block of calcium-permeable AMPA receptors.

Many properties of fast synaptic transmission in the brain are influenced by transmembrane AMPAR regulatory proteins (TARPs) that modulate the pharmacology and gating of AMPA-type glutamate receptors (AMPARs). Although much is known about TARP influence on AMPAR pharmacology and kinetics through their modulation of the extracellular ligand-binding domain (LBD), less is known about their regulation of the ion channel region. TARP-induced modifications in AMPAR channel behavior include increased single-channel conductance and weakened block of calcium-permeable AMPARs (CP-AMPARs) by endogenous intracellular polyamines. To investigate how TARPs modify ion flux and channel block, we examined the action of γ-2 (stargazin) on GluA1 and GluA4 CP-AMPARs. First, we compared the permeation of organic cations of different sizes. We found that γ-2 increased the permeability of several cations but not the estimated AMPAR pore size, suggesting that TARP-induced relief of polyamine block does not reflect altered pore diameter. Second, to determine whether residues in the TARP intracellular C-tail regulate polyamine block and channel conductance, we examined various γ-2 C-tail mutants. We identified the membrane proximal region of the C terminus as crucial for full TARP-attenuation of polyamine block, whereas complete deletion of the C-tail markedly enhanced the TARP-induced increase in channel conductance; thus, the TARP C-tail influences ion permeation. Third, we identified a site in the pore-lining region of the AMPAR, close to its Q/R site, that is crucial in determining the TARP-induced changes in single-channel conductance. This conserved residue represents a site of TARP action, independent of the AMPAR LBD.

α-Bungarotoxin labelling of AMPA receptor-associated TARPs in living neurons.

In mammalian central neurons AMPARs are clustered at glutamatergic synapses where they mediate fast excitatory transmission. In addition to four pore-forming subunits (GluA1-4), AMPARs contain auxiliary transmembrane AMPAR regulatory proteins (γ2, γ3, γ4, γ5, γ7 or γ8) whose incorporation can vary between neuron types, brain regions, and stages of development. As well as modulating the functional properties of AMPARs, these auxiliary subunits play a central role in AMPAR trafficking. Directly visualizing TARPs could therefore provide a valuable insight into mechanisms underlying these processes. Although antibodies are routinely used for the detection of surface proteins, our experience suggests anti-TARP antibodies are too bulky to access their target, possibly due to close interactions between the extracellular domains of TARP and AMPAR subunits. We therefore assessed the utility of a small monovalent probe - fluorescent α-bungarotoxin (α-Btx) - for TARP labelling in living neurons. We inserted the bungarotoxin binding site (BBS) within the extracellular domain of TARPs to enable their detection in cells exposed to fluorescent α-Btx. Focusing on the prototypical TARP γ2, we demonstrate that the small size of fluorescent α-Btx allows it to bind to the BBS-tagged TARP when associated with AMPARs. Importantly, labelled γ2 enhances AMPAR function in the same way as unmodified γ2. In living neurons, fluorescent α-Btx-labelled γ2 associates with AMPAR clusters at synapses. As a proof-of-principle, we employed our method to compare the surface trafficking of γ2 and γ7 in cerebellar stellate neurons. Our approach provides a simple way to visualize TARPs within AMPARs in living cells.

Setting the time course of inhibitory synaptic currents by mixing multiple GABA(A) receptor α subunit isoforms.

The kinetics of IPSCs influence many neuronal processes, such as the frequencies of oscillations and the duration of shunting inhibition. The subunit composition of recombinant GABA(A) receptors (GABA(A)Rs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABA(A)R subunits. In cerebellar stellate, thalamic relay, and main olfactory bulb (MOB) deep short-axon cells of Wistar rats, the only synaptic α subunit was α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τ(w)) of 4-6 ms. Nucleus reticularis thalami neurons expressed only α3 as the synaptic α subunit and exhibited slow (τ(w) = 28 ms), zolpidem-insensitive mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell, variability in synaptic α1/α3 ratios. This corresponded to large cell-to-cell variability in the decay (τ(w) = 3-30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1/α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABA(A)R subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.

Cornichons modify channel properties of recombinant and glial AMPA receptors.

Ionotropic glutamate receptors, which underlie a majority of excitatory synaptic transmission in the CNS, associate with transmembrane proteins that modify their intracellular trafficking and channel gating. Significant advances have been made in our understanding of AMPA-type glutamate receptor (AMPAR) regulation by transmembrane AMPAR regulatory proteins. Less is known about the functional influence of cornichons-unrelated AMPAR-interacting proteins, identified by proteomic analysis. Here we confirm that cornichon homologs 2 and 3 (CNIH-2 and CNIH-3), but not CNIH-1, slow the deactivation and desensitization of both GluA2-containing calcium-impermeable and GluA2-lacking calcium-permeable (CP) AMPARs expressed in tsA201 cells. CNIH-2 and -3 also enhanced the glutamate sensitivity, single-channel conductance, and calcium permeability of CP-AMPARs while decreasing their block by intracellular polyamines. We examined the potential effects of CNIHs on native AMPARs by recording from rat optic nerve oligodendrocyte precursor cells (OPCs), known to express a significant population of CP-AMPARs. These glial cells exhibited surface labeling with an anti-CNIH-2/3 antibody. Two features of their AMPAR-mediated currents-the relative efficacy of the partial agonist kainate (I(KA)/I(Glu) ratio 0.4) and a greater than fivefold potentiation of kainate responses by cyclothiazide-suggest AMPAR association with CNIHs. Additionally, overexpression of CNIH-3 in OPCs markedly slowed AMPAR desensitization. Together, our experiments support the view that CNIHs are capable of altering key properties of AMPARs and suggest that they may do so in glia.

An unexpected role for a glutamate receptor.

γ-Aminobutyric acid acts on a glutamate receptor, evoking synaptic plasticity.

Enhanced functional detection of synaptic calcium-permeable AMPA receptors using intracellular NASPM.

Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent channel block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits - including transmembrane AMPAR regulatory proteins, cornichons, and GSG1L - which are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here, we show that the inclusion of the spider toxin analog 1-naphthylacetyl spermine (NASPM) in the intracellular solution results in a complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, completely blocks outward synaptic currents. Thus, our results identify a functional measure of CP-AMPARs, that is unaffected by their auxiliary subunit content.

Profound desensitization by ambient GABA limits activation of δ-containing GABAA receptors during spillover.

High-affinity extrasynaptic GABA(A) receptors (GABA(A)Rs) are a prominent feature of cerebellar granule neurons and thalamic relay neurons. In both cell types, the presence of synaptic glomeruli would be expected to promote activation of these GABA(A)Rs, contributing to phasic spillover-mediated currents and tonic inhibition. However, the precise role of different receptor subtypes in these two phenomena is unclear. To address this question, we made recordings from neurons in acute brain slices from mice, and from tsA201 cells expressing recombinant GABA(A)Rs. We found that δ subunit-containing GABA(A)Rs of both cerebellar granule neurons and thalamic relay neurons of the lateral geniculate nucleus contributed to tonic conductance caused by ambient GABA but not to spillover-mediated currents. In the presence of a low "ambient" GABA concentration, recombinant "extrasynaptic" δ subunit-containing GABA(A)Rs exhibited profound desensitization, rendering them insensitive to brief synaptic- or spillover-like GABA transients. Together, our results demonstrate that phasic spillover and tonic inhibition reflect the activation of distinct receptor populations.

Probing TARP modulation of AMPA receptor conductance with polyamine toxins.

The properties of synaptic AMPA receptors (AMPARs) depend on their subunit composition and association with transmembrane AMPAR regulatory proteins (TARPs). Although both GluA2 incorporation and TARP association have been shown to influence AMPAR channel conductance, the manner in which different TARPs modulate the mean channel conductance of GluA2-containing AMPARs is unknown. Using ultrafast agonist application and nonstationary fluctuation analysis, we found that TARP subtypes differentially increase the mean channel conductance, but not the peak open probability, of recombinant GluA2-containing AMPARs. TARP γ-8, in particular, enhances mean channel conductance to a greater degree than γ-2, γ-3, or γ-4. We then examined the action of a use-dependent antagonist of GluA2-containing AMPARs, philanthotoxin-74 (PhTx-74), on recombinant AMPARs and on GluA2-containing AMPARs in cerebellar granule neurons from stargazer mice transfected with TARPs. We found that the rate and extent of channel block varies with TARP subtype, in a manner that correlates linearly with mean channel conductance. Furthermore, block of GluA2-containing AMPARs by polyamine toxins varied depending on whether channels were activated by the full agonist glutamate or the partial agonist kainate, consistent with conductance state-dependent block. Block of GluA2-lacking AMPARs by PhTx-433 is also modulated by TARP association and is a function of agonist efficacy. Our data indicate that channel block by polyamine toxins is sensitive to the mean channel conductance of AMPARs, which varies with TARP subtype and agonist efficacy. Furthermore, our results illustrate the utility of polyamine toxins as sensitive probes of AMPAR channel conductance and suggest the possibility that TARPs may influence their channel properties by selectively stabilizing specific channel conformations, rather than altering the pore structure.

TARP-associated AMPA receptors display an increased maximum channel conductance and multiple kinetically distinct open states.

Fast excitatory synaptic transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs), whose biophysical properties are dramatically modulated by the presence of transmembrane AMPAR regulatory proteins (TARPs). To help construct a kinetic model that will realistically describe native AMPAR/TARP function, we have examined the single-channel properties of homomeric GluA1 AMPARs in combination with the TARPs, γ-2, γ-4 and γ-5. In a saturating concentration of agonist, each of these AMPAR/TARP combinations gave rise to single-channel currents with multiple conductance levels that appeared intrinsic to the receptor-channel complex, and showed long-lived subconductance states. The open time and burst length distributions of the receptor complexes displayed multiple dwell-time components. In the case of γ-2- and γ-4-associated receptors, these distributions included a long-lived component lasting tens of milliseconds that was absent from both GluA1 alone and γ-5-associated receptors. The open time distributions for each conductance level required two dwell-time components, indicating that at each conductance level the channel occupies a minimum of two kinetically distinct open states. We have explored how these data place novel constraints on possible kinetic models of TARP-associated AMPARs that may be used to define AMPAR-mediated synaptic transmission.

Stargazin attenuates intracellular polyamine block of calcium-permeable AMPA receptors.

Endogenous polyamines profoundly affect the activity of various ion channels, including that of calcium-permeable AMPA-type glutamate receptors (CP-AMPARs). Here we show that stargazin, a transmembrane AMPAR regulatory protein (TARP) known to influence transport, gating and desensitization of AMPARs, greatly reduces block of CP-AMPARs by intracellular polyamines. By decreasing CP-AMPAR affinity for cytoplasmic polyamines, stargazin enhances the charge transfer following single glutamate applications and eliminates the frequency-dependent facilitation seen with repeated applications. In cerebellar stellate cells, which express both synaptic CP-AMPARs and stargazin, we found that the rectification and unitary conductance of channels underlying excitatory postsynaptic currents were matched by those of recombinant AMPARs only when the latter were associated with stargazin. Taken together, our observations establish modulatory actions of stargazin that are specific to CP-AMPARs, and suggest that during synaptic transmission the activity of such receptors, and thus calcium influx, is fundamentally changed by TARPs.

From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors.

In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the gamma2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase-induced swapping of the gamma2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.

Transient developmental imbalance of cortical interneuron subtypes presages long-term changes in behavior.

Cortical GABAergic interneurons are generated in large numbers in the ganglionic eminences and migrate into the cerebral cortex during embryogenesis. At early postnatal stages, during neuronal circuit maturation, autonomous and activity-dependent mechanisms operate within the cortex to adjust cell numbers by eliminating naturally occurring neuron excess. Here, we show that when cortical interneurons are generated in aberrantly high numbers-due to a defect in precursor cell proliferation during embryogenesis-extra parvalbumin interneurons persist in the postnatal mouse cortex during critical periods of cortical network maturation. Even though cell numbers are subsequently normalized, behavioral abnormalities remain in adulthood. This suggests that timely clearance of excess cortical interneurons is critical for correct functional maturation of circuits that drive adult behavior.

Barrett's metaplasia: molecular mechanisms and nutritional influences.

Barrett's metaplasia is discussed in the context of a general theory for the formation of metaplasias based on developmental biology. The phenotype of a particular tissue type becomes established during embryonic development by the expression of a specific set of transcription factors. If this combination becomes altered, then the tissue type can be altered. Such events may occur by mutation or by environmental effects on gene expression, normally within the stem cell population of the tissue. A macroscopic patch of metaplastic tissue will arise only if the new gene activity state is self-sustaining in the absence of its original causes, and if the new tissue type can outgrow the parent tissue type. An important candidate gene for the causation of Barrett's metaplasia is Cdx2 (Caudal-type homeobox 2). In normal development, this is expressed in the future intestine, but not the future foregut. Mouse knockout studies have shown that it is needed for intestinal development, and that its loss from adult intestine can lead to squamous transformations. It is also expressed in Barrett's metaplasia and can be activated in oesophageal cell cultures by treatment with bile acids. We have investigated the ability of Cdx2 to bring about intestinal transformations in oesophageal epithelium. Our results show that Cdx2 can activate a programme of intestinal gene expression when overexpressed in HET-1A cells, or in fetal epithelium, but not in the adult epithelium. This suggests that Cdx2, although necessary for formation of intestinal tissue, is not sufficient to provoke Barrett's metaplasia in adult life and that overexpression of additional transcription factors is necessary. In terms of diet and nutrition, there is a known association of Barrett's metaplasia with obesity. This may work through an increased risk of gastro-oesophageal reflux. Acid and bile are known to activate Cdx2 expression in oesophageal cells. It may also increase circulating levels of TNFalpha (tumour necrosis factor alpha), which activates Cdx2. In addition, there may be effects of diet on the composition of the bile.