Type I TARPs promote dendritic growth of early postnatal neocortical pyramidal cells in organotypic cultures.

The ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptors (AMPARs) have been implicated in the establishment of dendritic architecture. The transmembrane AMPA receptor regulatory proteins (TARPs) regulate AMPAR function and trafficking into synaptic membranes. In the current study, we employ type I and type II TARPs to modulate expression levels and function of endogenous AMPARs and investigate in organotypic cultures (OTCs) of rat occipital cortex whether this influences neuronal differentiation. Our results show that in early development [5-10 days in vitro (DIV)] only the type I TARP γ-8 promotes pyramidal cell dendritic growth by increasing spontaneous calcium amplitude and GluA2/3 expression in soma and dendrites. Later in development (10-15 DIV), the type I TARPs γ-2, γ-3 and γ-8 promote dendritic growth, whereas γ-4 reduced dendritic growth. The type II TARPs failed to alter dendritic morphology. The TARP-induced dendritic growth was restricted to the apical dendrites of pyramidal cells and it did not affect interneurons. Moreover, we studied the effects of short hairpin RNA-induced knockdown of endogenous γ-8 and showed a reduction of dendritic complexity and amplitudes of spontaneous calcium transients. In addition, the cytoplasmic tail (CT) of γ-8 was required for dendritic growth. Single-cell calcium imaging showed that the γ-8 CT domain increases amplitude but not frequency of calcium transients, suggesting a regulatory mechanism involving the γ-8 CT domain in the postsynaptic compartment. Indeed, the effect of γ-8 overexpression was reversed by APV, indicating a contribution of NMDA receptors. Our results suggest that selected type I TARPs influence activity-dependent dendritogenesis of immature pyramidal neurons.

C-terminal domains of transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor regulatory proteins not only facilitate trafficking but are major modulators of AMPA receptor function.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors are essential players in fast synaptic transmission in the vertebrate central nervous system. Their synaptic delivery and localization as well as their electrophysiological properties are regulated by transmembrane AMPA receptor regulatory proteins (TARPs). However, the exact mechanisms of how the four originally designated TARPs (gamma2, gamma3, gamma4, and gamma8) modulate AMPA receptor function remain largely unknown. Previous studies suggested the C-terminal domain (CTD) of gamma2 to mediate increased trafficking and reduced desensitization of AMPA receptors. As it remained unclear whether these findings extend to other TARPs, we set out to investigate and compare the role of the CTDs of the four original TARPs in AMPA receptor modulation. To address this issue, we replaced the TARP CTDs with the CTD of the homologous subunit gamma1, a voltage-dependent calcium channel subunit expressed in skeletal muscle that lacks TARP properties. We analyzed the impact of the resulting chimeras on GluR1 functional properties in Xenopus oocytes and HEK293 cells. Interestingly, the CTDs of all TARPs not only modulate the extent and kinetics of desensitization but also modulate agonist potencies of AMPA receptors. Furthermore, the CTDs are required for TARP-induced modulation of AMPA receptor gating, including conversion of antagonists to partial agonists and constitutive channel openings. Strikingly, we found a special role of the cytoplasmic tail of gamma4, suggesting that the underlying mechanisms of modulation of AMPA receptor function are different among the TARPs. We propose that the intracellularly located CTD is the origin of TARP-specific functional modulation and not merely a facilitator of trafficking.

The glutamate receptor subunit delta2 is capable of gating its intrinsic ion channel as revealed by ligand binding domain transplantation.

The family of ionotropic glutamate receptors includes 2 subunits, delta1 and delta2, the physiological relevance of which remains poorly understood. Both are nonfunctional in heterologous expression systems, although the isolated, crystallized ligand binding domain (LBD) of delta2 is capable of binding D-serine. To investigate these seemingly contradictory observations we tested whether delta receptors can be ligand gated at all. We used a strategy that replaced the native LBD of delta2 by a proven glutamate-binding LBD. Test transplantations between alpha-amino-3-hydroxy-5-methylisoxazole propionate (AMPA) and kainate receptors (GluR1 and GluR6, respectively) showed that this approach can produce functional chimeras even if only one part of the bipartite LBD is swapped. Upon outfitting delta2 with the LBD of GluR6, the chimera formed glutamate-gated ion channels with low Ca(2+) permeability and unique rectification properties. Ligand-induced conformational changes can thus gate delta2, suggesting that the LBD of this receptor works fundamentally differently from that of other ionotropic glutamate receptors.

Different structural requirements for functional ion pore transplantation suggest different gating mechanisms of NMDA and kainate receptors.

Although considerable progress has been made in characterizing the physiological function of the high-affinity kainate (KA) receptor subunits KA1 and KA2, no homomeric ion channel function has been shown. An ion channel transplantation approach was employed in this study to directly test if homomerically expressed KA1 and KA2 pore domains are capable of conducting currents. Transplantation of the ion pore of KA1 or KA2 into GluR6 generated perfectly functional ion channels that allowed characterization of those electrophysiological and pharmacological properties that are determined exclusively by the ion pore of KA1 or KA2. This demonstrates for the first time that KA1 and KA2 ion pore domains are intrinsically capable of conducting ions even in homomeric pore assemblies. NMDA receptors, similar to KA1- or KA2-containing receptors, function only as heteromeric complexes. They are composed of NR1 and NR2 subunits, which both are non-functional when expressed homomerically. In contrast to NR1, the homomeric NR2B ion pore failed to translate ligand binding into pore opening when transplanted into GluR6. Similarly, heteromeric coexpression of the ion channel domains of both NR1 and NR2 inserted into GluR6 failed to produce functional channels. Therefore, we conclude that the mechanism underlying the ion channel opening in the obligatorily heterotetrameric NMDA receptors differs significantly from that in the facultatively heterotetrameric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and KA receptors.

The transmembrane AMPA receptor regulatory protein gamma 4 is a more effective modulator of AMPA receptor function than stargazin (gamma 2).

AMPA receptors mediate the majority of the fast excitatory synaptic transmission in the brain. A family of recently described auxiliary proteins, the transmembrane AMPA receptor regulatory proteins (TARPs) gamma2, gamma3, gamma4, and gamma8, have been shown to modulate the trafficking of receptors to the plasma membrane as well as electrophysiological key properties. Most studies published to date focus exclusively on gamma2 (stargazin), neglecting the other three members of the TARP family. Here, we analyzed the modulation of electrophysiological properties of AMPA receptors by gamma4 and compare it with gamma2, using heterologous coexpression in human embryonic kidney 293 cells. We show for the first time that gamma4, a previously poorly examined TARP, modulates the desensitization properties of AMPA receptors significantly stronger than gamma2 does. In contrast, other properties such as kainate efficacy and current-voltage relationships are modulated in a similar way by both of these TARPs. From these TARP-specific effects, we propose an interaction mechanism between AMPA receptors and TARPs and address the physiological relevance of gamma4 and its regulatory effects, particularly on AMPA receptor desensitization properties, to developmental and regulatory processes in the brain.

Stargazin interaction with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors is critically dependent on the amino acid at the narrow constriction of the ion channel.

The subunit GluR2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subfamily of ionotropic glutamate receptors (GluRs) features a single amino acid at the narrow constriction of the pore loop that is altered from glutamine to arginine by RNA editing. This so-called Q/R site has been shown to play an important role in the determination of the electrophysiological properties of AMPA receptor complexes as well as of trafficking to the plasma membrane. The protein stargazin has also been shown to modulate electrophysiological properties and trafficking to the plasma membrane of AMPA receptors. In this study we examined via a series of mutants of the Q/R site of the AMPA receptor GluR1 whether the amino acid at this position has any influence on the modulatory effects mediated by stargazin. To this end, we analyzed current responses of Q/R site mutants upon application of glutamate and kainate and determined the amount of mutant receptor protein in the plasma membrane in Xenopus oocytes. Desensitization kinetics of several mutants were analyzed in HEK293 cells. We found that the stargazin-mediated decrease in receptor desensitization, the slowing of desensitization kinetics, and the kainate efficacy were all dependent on the amino acid at the Q/R site, whereas the stargazin-mediated increase in trafficking toward the plasma membrane remained independent of this amino acid. We propose that the Q/R site modulates the interaction of stargazin with the transmembrane domains of AMPA receptors via an allosteric mechanism and that this modulation leads to the observed differences in the electrophysiological properties of the receptor.

Electrophysiological properties of AMPA receptors are differentially modulated depending on the associated member of the TARP family.

The family of AMPA receptors is encoded by four genes that are differentially spliced to result in the flip or flop versions of the four subunits GluR1 to GluR4. GluR2 is further modified at the so-called Q/R site by posttranscriptional RNA editing. Delivery of AMPA receptors to the plasma membrane and synaptic trafficking are controlled by transmembrane AMPA receptor regulatory proteins (TARPs). Additionally, TARPs influence essential electrophysiological properties of AMPA receptor channels such as desensitization and agonist efficacies. Here, we compare the influence of all known TARPs (gamma2, gamma3, gamma4, and gamma8) on agonist-induced currents of the four AMPA receptor subunits, including flip and flop splice variants and editing variants. We show that, although agonist-induced currents of all homomeric AMPA receptor subunits as well as all heteromeric combinations tested are significantly potentiated when coexpressed with members of the TARP family in Xenopus laevis oocytes, the extent of TARP-mediated increase in agonist-induced responses is highly dependent on both the AMPA receptor subunit and the coexpressed TARP. Moreover, we demonstrate that the splice variant of the AMPA receptor plays a key role in determining the modulation of electrophysiological properties by associated TARPs. We furthermore present evidence that individual TARP-AMPA receptor interactions control the degree of desensitization of AMPA receptors. Consequently, because of their subunit-specific impact on the electrophysiological properties, TARPs play a major role as modulatory subunits of AMPA receptors and thus contribute to the functional diversity of AMPA receptors encountered in the CNS.