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.

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.

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.

The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking.

Accumulation of AMPA receptors at synapses is a fundamental feature of glutamatergic synaptic transmission. Stargazin, a member of the TARP family, is an AMPAR auxiliary subunit allowing interaction of the receptor with scaffold proteins of the postsynaptic density, such as PSD-95. How PSD-95 and Stargazin regulate AMPAR number in synaptic membranes remains elusive. We show, using single quantum dot and FRAP imaging in live hippocampal neurons, that exchange of AMPAR by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 AMPAR subunit C terminus. Disruption of interactions between Stargazin and PSD-95 strongly increases AMPAR surface diffusion, preventing AMPAR accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin-PSD-95 interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density.

A role of TARPs in the expression and plasticity of calcium-permeable AMPARs: evidence from cerebellar neurons and glia.

The inclusion of GluA2 subunits has a profound impact on the channel properties of AMPA receptors (AMPARs), in particular rendering them impermeable to calcium. While GluA2-containing AMPARs are the most abundant in the central nervous system, GluA2-lacking calcium-permeable AMPARs are also expressed in wide variety of neurons and glia. Accumulating evidence suggests that the dynamic control of the GluA2 content of AMPARs plays a critical role in development, synaptic plasticity, and diverse neurological conditions ranging from ischemia-induced brain damage to drug addiction. It is thus important to understand the molecular mechanisms involved in regulating the balance of AMPAR subtypes, particularly the role of their co-assembled auxiliary subunits. The discovery of transmembrane AMPAR regulatory proteins (TARPs), initially within the cerebellum, has transformed the field of AMPAR research. It is now clear that these auxiliary subunits play a key role in multiple aspects of AMPAR trafficking and function in the brain. Yet, their precise role in AMPAR subtype-specific regulation has only recently received particular attention. Here we review recent findings on the differential regulation of calcium-permeable (CP-) and -impermeable (CI-) AMPARs in cerebellar neurons and glial cells, and discuss the critical involvement of TARPs in this process. This article is part of the Special Issue entitled 'Glutamate Receptor-Dependent Synaptic Plasticity'.

Channel properties reveal differential expression of TARPed and TARPless AMPARs in stargazer neurons.

Dynamic regulation of calcium-permeable AMPA receptors (CP-AMPARs) is important for normal synaptic transmission, plasticity and pathological changes. Although the involvement of transmembrane AMPAR regulatory proteins (TARPs) in trafficking of calcium-impermeable AMPARs (CI-AMPARs) has been extensively studied, their role in the surface expression and function of CP-AMPARs remains unclear. We examined AMPAR-mediated currents in cerebellar stellate cells from stargazer mice, which lack the prototypical TARP stargazin (g-2). We found a marked increase in the contribution of CP-AMPARs to synaptic responses, indicating that, unlike CI-AMPARs, these can localize at synapses in the absence of g-2. In contrast with CP-AMPARs in extrasynaptic regions, synaptic CP-AMPARs displayed an unexpectedly low channel conductance and strong block by intracellular spermine, suggesting that they were 'TARPless'. As a proof of principle that TARP association is not an absolute requirement for AMPAR clustering at synapses, miniature excitatory postsynaptic currents mediated by TARPless AMPARs were readily detected in stargazer granule cells following knockdown of their only other TARP, g-7.

Direct imaging of lateral movements of AMPA receptors inside synapses.

Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single-molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra-synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra-synaptic localization occurs through regulation of receptor diffusion inside synapses.