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 specific to CP-AMPARs, and suggest that during synaptic transmission the activity of such receptors, and thus calcium influx, is fundamentally changed by TARPs.

Although the properties and trafficking of AMPA-type glutamate receptors (AMPARs) depend critically on associated transmembrane AMPAR regulatory proteins (TARPs) such as stargazin (γ-2), no TARP has been described that can specifically regulate the important class of calciumpermeable (CP-) AMPARs. We examined the stargazin-related protein γ-5, which is highly expressed in Bergmann glia, a cell type possessing only CP-AMPARs. γ-5 was previously thought not to be a TARP, and it has been widely used as a negative control. Here we find that, contrary to expectation, γ-5 acts as a TARP and serves this role in Bergmann glia. Whereas, γ-5 interacts with all AMPAR subunits, and modifies their behavior to varying extents, its main effect is to regulate the function of AMPAR subunit combinations that lack short-form subunits, which constitute predominantly CP-AMPARs. Our results suggest an important role γ-5 in regulating the functional contribution of CP-AMPARs.

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 calciumimpermeable 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.

EuroEPINOMICS (European Science Foundation through national funding organisations), Epicure and EpiPGX (Sixth Framework Programme and Seventh Framework Programme of the European Commission), Research Unit FOR2715 (German Research Foundation and Luxembourg National Research Fund).

SummaryFast excitatory transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs) associated with transmembrane AMPAR regulatory proteins (TARPs). At the high glutamate concentrations typically seen during synaptic transmission, TARPs slow receptor desensitization and enhance mean channel conductance. However, their influence on channels gated by low glutamate concentrations, as encountered during delayed transmitter clearance or synaptic spillover, is poorly understood. We report here that TARP γ-2 reduces the ability of low glutamate concentrations to cause AMPAR desensitization and enhances channel gating at low glutamate occupancy. Simulations show that, by shifting the balance between AMPAR activation and desensitization, TARPs can markedly facilitate the transduction of spillover-mediated synaptic signaling. Furthermore, the dual effects of TARPs can account for biphasic steady-state glutamate concentration-response curves—a phenomenon termed “autoinactivation,” previously thought to reflect desensitization-mediated AMPAR/TARP dissociation.

Heterogeneity among AMPA receptor (AMPAR) subtypes is thought to be one of the key postsynaptic factors giving rise to diversity in excitatory synaptic signaling in the CNS. Recently, compelling evidence has emerged that ancillary AMPAR subunits—the so-called transmembrane AMPA receptor regulatory proteins (TARPs)—also play a vital role in influencing the variety of postsynaptic signaling. This TARP family of molecules controls both trafficking and functional properties of AMPARs at most, if not all, excitatory central synapses. Furthermore, individual TARPs differ in their effects on the biophysical and pharmacological properties of AMPARs. The critical importance of TARPs in synaptic transmission was first revealed in experiments on cerebellar granule cells from stargazer mice. These lack the prototypic TARP stargazin, present in granule cells from wild-type animals, and consequently lack synaptic transmission at the mossy fibre-to-granule cell synapse. Subsequent work has identified many other members of the stargazin family which act as functional TARPs. It has also provided valuable information about specific TARPs present in many central neurons. Because much of the initial work on TARPs was carried out on stargazer granule cells, the important functional properties of TARPs present throughout the cerebellum have received particular attention. Here we discuss some of these recent findings in relation to the main TARPs and the AMPAR subunits identified in cerebellar neurons and glia.

SummaryRegulation of calcium-permeable AMPA receptors (CP-AMPARs) is critical in normal synaptic function and neurological disease states. While transmembrane AMPAR regulatory proteins (TARPs) such as stargazin (γ-2) modulate the properties of calcium-impermeable (CI-) AMPARs and promote their synaptic targeting, the TARP-specific rules governing CP-AMPAR synaptic trafficking remain unclear. We used RNA interference to manipulate AMPAR subunit- and TARP expression in γ-2-lacking stargazer cerebellar granule cells – the classic model of TARP deficiency. We found that TARP γ-7 selectively enhanced synaptic expression of CP-AMPARs and suppressed CI-AMPARs, identifying a pivotal role of γ-7 in regulating the prevalence of CP-AMPARs. In the absence of associated TARPs, although their properties were altered, both CP- and CI-AMPARs were able to localize at synapses and mediate transmission. Finally, our results establish that TARPed synaptic receptors in granule cells require both γ-2 and γ-7, and reveal an unexpected basis for the loss of AMPAR-mediated transmission in stargazer mice.

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 ␥-2 (stargazin) on GluA1 and GluA4 CP-AMPARs. First, we compared the permeation of organic cations of different sizes. We found that ␥-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 ␥-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.