Glutamate receptors of the AMPA subtype (AMPARs) mediate fast synaptic transmission in the brain. These ionotropic receptors rely on auxiliary subunits known as transmembrane AMPAR regulatory proteins (TARPs) for both trafficking and gating. Recently, a second family of AMPAR binding proteins, referred to as cornichons, were identified and also proposed to function as auxiliary subunits. Cornichons are transmembrane proteins that modulate AMPAR function in expression systems much like TARPs. In the present study we compare the role of cornichons in controlling AMPA receptor function in neurons and HEK cells to that of TARPs. Cornichons mimic some, but not all, of the actions of TARPs in HEK cells; their role in neurons, however, is more limited. Although expressed cornichons can affect the trafficking of AMPARs, they were not detected on the surface of neurons and failed to alter the kinetics of endogenous AMPARs. This neuronal role is more consistent with that of an endoplasmic reticulum (ER) chaperone rather than a bona fide auxiliary subunit.stargazin | synapse | auxiliary subunit | hippocampus

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 ␣-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. Ligandinduced conformational changes can thus gate delta2, suggesting that the LBD of this receptor works fundamentally differently from that of other ionotropic glutamate receptors.orphan receptor ͉ AMPA receptor ͉ kainate receptor ͉ chimeric receptors ͉ ligand binding domain

The two delta receptor subunits remain the most puzzling enigma within the ionotropic glutamate receptor family. Despite the recent elucidation of the ligand-binding domain structure of delta2, many fundamental questions with regard to the subunits' mechanism of function still remain unanswered. Of necessity, the majority of studies on delta receptors focused on the metabotropic function of delta2, since electrophysiological approaches to date are limited to the characterization of spontaneous currents through the delta2-lurcher mutant. Indeed, accumulated evidence primarily from delta2-deficient transgenic mice suggest that major physiological roles of delta2 are mediated via metabotropic signaling by the subunit's C terminus. Why then would the subunits retain a conserved ion channel domain if they do not form functional ion channels? Any progress with regard to ionotropic function of the two delta subunits has been hampered by their largely unknown pharmacology. Even now that a pharmacological profile for delta2 is being established on the basis of the ligand-binding domain structure, wild-type delta2 channels in heterologous expression systems stay closed in the presence of molecules that have been demonstrated to bind to the receptor's ligand-binding domain. In this paper, we review the current knowledge of delta subunits focusing on the disputed ionotropic function.

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.Key words: glutamate; AMPA receptor; gating; lurcher; linker; desensitization IntroductionIonotropic glutamate receptors (GluRs) are responsible for the majority of excitatory neurotransmission in the mammalian brain . Each GluR subunit harbors an extracellular N terminus, an intracellular C terminus, three transmembrane domains (TMDs; A-C), and a hairpin loop between TMDs A and B (see Fig. 1 B). TMD B and the hairpin loop are the major channel-lining domains and together with TMD A are inserted between two discontinuous stretches of sequence, S1 and S2, which make up the ligand-binding domain (LBD).Gating in GluRs encompasses the conformational change induced by ligand binding and its subsequent translation into pore opening. This movement is propagated via three short linkers of 9 -17 aa that connect the LBD with the TMDs, presumably forming the outer vestibule of the ion channel (Beck et al., 1999). Inclusion of these linkers in the crystal structure of the LBD was precluded by their flexibility (Armstrong et al., 1998), and structural determinants of gating that reside within them still remain completely unknown. So far it has only been reported that mutations in the linker preceding TMD A (linker A) (Stern-Bach et al., 1998), as well as a point mutation in the linker following TMD B (linker B), attenuate AMPA receptor (AMPAR) desensitization .We transplanted all th...

The subunit GluR2 of the ␣-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.