Markus Werner

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 (γ2, γ3, γ4, and γ8) 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.

Human Trace Amine-Associated Receptor TAAR5 Can Be Activated by Trimethylamine

In addition to the canonical olfactory receptors, TAARs were currently suggested to be a second class of chemosensory receptors in the olfactory epithelium of vertebrates. In contrast to several deorphanized murine TAARs, agonists for the intact human TAAR genes 2, 5, 6, 8 and 9 that are potentially expressed in the human olfactory epithelium have not been determined so far. Moreover, the physiological relevance of TAARs still remains elusive. We present the first successful functional expression of a human TAAR and agonists of human TAAR5. We performed a ligand screening using recombinantly expressed human TAAR5 in HANA3A cells and Xenopus laevis oocytes. In order to measure receptor activity, we used a cAMP-dependent reporter gene assay and two-electrode voltage clamp technique. As a result, human TAAR5 can be activated in a concentration-dependent manner by trimethylamine and with less efficacy by dimethylethylamine. It could neither be activated by any other of the tested single amines with a related chemical structure (42 in total), nor by any of the tested odorant mixtures. The hypothesis that Single Nucleotide Polymorphisms (SNP) within the reading frame of an olfactory receptor gene can cause a specific anosmia, formed the basis for clarifying the question, if anosmia for trimethylamine is caused by a SNP in a TAAR coding sequence. All functional human TAAR gene reading frames of subjects with specific anosmia for trimethylamine were amplified and products analyzed regarding SNP distribution. We demonstrated that the observed specific anosmia for trimethylamine is not correlated with a SNP in the coding sequence of one of the putatively functional human TAAR genes.

Comparative analysis of the pharmacology of GluR1 in complex with transmembrane AMPA receptor regulatory proteins γ2, γ3, γ4, and γ8

AMPA receptors (AMPARs) mediate the majority of fast synaptic transmission in the CNS of vertebrates. They are believed to be associated with members of the transmembrane AMPA receptor regulatory protein (TARP) family. TARPs mediate the delivery of AMPA receptors to the plasma membrane and mediate their synaptic trafficking. Moreover, TARPs modulate essential electrophysiological properties of AMPA receptors. Here, we compare the influence of rat TARPs (gamma2, gamma3, gamma4, and gamma8) on pharmacological properties of rat GluR1(Q)flip. We show that agonist potencies are increased by all TARPs, but to individually different extents. On the other hand, all TARPs increase agonist potencies at the virtually non-desensitizing mutant GluR1-L479Y almost identically. Comparison of the influence of individual TARPs on relative agonist efficacies confirmed that the TARPs can be functionally subdivided into two subgroups, one consisting of gamma2 and gamma3 and one consisting of gamma4 and gamma8. Surprisingly, we found that TARPs convert certain AMPA receptor antagonists to agonists. The potency of one of these converted antagonists is dependent on the particular TARP. Moreover, TARPs (except gamma4) reduce the ion channel block by the synthetic Joro spider toxin analog 1-naphthylacetyl spermine (NASP). In addition, TARPs increase the permeability of the receptor to calcium, indicating that TARPs directly modulate important ion pore properties. In summary, the data presented herein will illustrate and help to understand the previously unexpected complexities of modulation of AMPA receptor pharmacological properties by TARPs.

The Transmembrane AMPA Receptor Regulatory Protein γ4 Is a More Effective Modulator of AMPA Receptor Function than Stargazin (γ2)

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) γ2, γ3, γ4, and γ8, 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 γ2 (stargazin), neglecting the other three members of the TARP family. Here, we analyzed the modulation of electrophysiological properties of AMPA receptors by γ4 and compare it with γ2, using heterologous coexpression in human embryonic kidney 293 cells. We show for the first time that γ4, a previously poorly examined TARP, modulates the desensitization properties of AMPA receptors significantly stronger than γ2 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 γ4 and its regulatory effects, particularly on AMPA receptor desensitization properties, to developmental and regulatory processes in the brain.

Stargazin Interaction with α-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 α-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.

Bi-directional control of motor neuron dendrite remodeling by the calcium permeability of AMPA receptors

Motor neurons express particularly high levels of the AMPA receptor subunit GluR1(Q)flip (GluR1(Q)i) during the period in early postnatal life when their dendritic tree grows and becomes more branched. To investigate how GluR1-containing AMPA receptors contribute to dendrite morphogenesis, we characterized a mutant form of GluR1 (containing a histidine in the Q/R editing site) with unique electrophysiological properties. Most notably, AMPA receptors assembled from GluR1(H)i display less calcium permeability than AMPA receptors assembled from GluR1(Q)i. Expression of GluR1(Q)i in vivo or in vitro led to an increase in dendrite branching with no net change in the overall tree size while GluR1(H)i led to a loss of branches and a net reduction in overall tree size. GluR1(H)i-dependent dendrite atrophy is mediated by protein phosphatase 2B. The results suggest that the electrophysiological properties of cell surface AMPA receptors, specifically their permeability to calcium, can be a central determinant of whether the dendrites undergo activity-dependent branching or atrophy.

Role of GluR1 in activity-dependent motor system development

Activity-dependent specification of neuronal architecture during early postnatal life is essential for refining the precision of communication between neurons. In the spinal cord under normal circumstances, the AMPA receptor subunit GluR1 is expressed at high levels by motor neurons and surrounding interneurons during this critical developmental period, although the role it plays in circuit formation and locomotor behavior is unknown. Here, we show that GluR1 promotes dendrite growth in a non-cell-autonomous manner in vitro and in vivo. The mal-development of motor neuron dendrites is associated with changes in the pattern of interneuronal connectivity within the segmental spinal cord and defects in strength and endurance. Transgenic expression of GluR1 in adult motor neurons leads to dendrite remodeling and supernormal locomotor function. GluR1 expression by neurons within the segmental spinal cord plays an essential role in formation of the neural network that underlies normal motor behavior.

A Domain Linking the AMPA Receptor Agonist Binding Site to the Ion Pore Controls Gating and Causes lurcher Properties when Mutated

Ionotropic, AMPA-type glutamate receptors (GluRs) critically shape excitatory synaptic signals in the CNS. Ligand binding induces conformational changes in the glutamate-binding domain of the receptors that are converted into opening of the channel pore via three short linker sequences, a process referred to as gating. Although crystallization of the glutamate-binding domain and structural models of the ion pore advanced our understanding of ligand-binding dynamics and pore movements, the allosteric coupling of both events by the short linkers has not been described in detail. To study the role of the linkers in gating GluR1, we transplanted them between different GluRs and examined the electrophysiological properties of the resulting chimeric receptors in Xenopus laevis oocytes and HEK293 cells. We found that all three linkers decisively affect receptor functionality, agonist potency, and desensitization. One linker chimera was nondesensitizing and exhibited strongly increased agonist potencies, while fluxing ions even in the absence of agonist, similar to properties reported for the GluR1 lurcher mutation. Combining this new lurcher-like linker chimera with the original lurcher mutation allowed us to reassess the effect of lurcher on GluR1 gating properties. The observed differential but interdependent influence of linker and lurcher mutations on receptor properties suggests that the linkers are part of a fine-tuned structural element that normally stabilizes the closed ion pore. We propose that lurcher-like mutations act by disrupting this element such that ligand-induced conformational changes are not necessarily required to gate the channel.

Functional excitatory GABAA receptors precede ionotropic glutamate receptors in radial glia-like neural stem cells.

The involvement of neurotransmission in neuronal development is a generally accepted concept. Nevertheless, the precise regulation of neurotransmitter receptor expression is still unclear. To investigate the expression profiles of the most important ionotropic neurotransmitter receptors, namely GABA(A) receptors (GABA(A)Rs), NMDA receptors (NMDARs), and AMPA receptors (AMPARs), quantitative RT-PCR, immunoblot analysis and patch clamp studies were performed in in vitro-generated neural stem cells (NSCs). This clearly defined cell line is closely related to radial glia cells, the stem cells in the neonate brain. We found functional GABA(A)Rs of the subunit composition alpha2, beta3, and gamma1 to be expressed. Unexpectedly, functional ionotropic glutamate receptors were absent. However, NSCs expressed the NMDAR subunits NR2A and NR3A, and the AMPAR subunit GluR4 at the protein level, and GluR3 at the mRNA level. The overexpression of functional NMDARs in NSCs led to an increased mRNA level of AMPAR subunits, indicating a role in synaptogenesis. Early neuronal markers remained unchanged. These data extend our knowledge about ionotropic neurotransmitter receptor expression during neuronal development and will aid further investigations on activity-dependent neurogenesis.

Revisiting the postulated "unitary glutamate receptor": electrophysiological and pharmacological analysis in two heterologous expression systems fails to detect evidence for its existence.

Several years ago evidence for a so-called “unitary glutamate receptor” was published. This unique type of glutamate receptor was reported to be activated by the traditional agonists of all three major glutamate receptor subfamilies [i.e., α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-d-aspartate (NMDA)] in a glycine-dependent as well as magnesium-blockable manner and was reported to consist of an NR1 subunit coexpressed with the kainate binding protein (KBP) from Xenopus laevis, XenU1. To re-examine the existence of such a receptor, we cloned two splice variants of the X. laevis NMDA receptor subunit NR1, XenNR1-4a and XenNR1-4b, and expressed them in X. laevis oocytes as well as in human embryonic kidney (HEK) 293 cells, either alone or with the X. laevis KBP subunit XenU1. In addition, we coexpressed XenU1 separately with all eight splice variants of the rat NR1 subunit. In no case did we see evidence of a unitary glutamate receptor pharmacology. In HEK293 cells, we did not get receptor response unless an NR2 subunit was coexpressed. In X. laevis oocytes, we did observe responses to glutamate/glycine as well as small responses to glycine alone, but these were independent of coexpressed XenU1. Neither AMPA nor kainate ever elicited significant responses. Because we verified that XenU1 is expressed and inserted into the plasma membrane of HEK293 cells, we conclude that XenU1 and NR1 do not form the postulated unitary glutamate receptor. Furthermore, successful amplification of a fragment of a X. laevis NR2 subunit indicates that X. laevis uses NR2 subunits and not XenU1 to form heteromeric complexes with NR1.