The Transmembrane Domain C of AMPA Receptors is Critically Involved in Receptor Function and Modulation

Ionotropic glutamate receptors are major players in synaptic transmission and are critically involved in many cognitive events. Although receptors of different subfamilies serve different functions, they all show a conserved domain topology. For most of these domains, structure–function relationships have been established and are well understood. However, up to date the role of the transmembrane domain C in receptor function has been investigated only poorly. We have constructed a series of receptor chimeras and point mutants designed to shed light on the structural and/or functional importance of this domain. We here present evidence that the role of transmembrane domain C exceeds that of a mere scaffolding domain and that several amino acid residues located within the domain are crucial for receptor gating and desensitization. Furthermore, our data suggest that the domain may be involved in receptor interaction with transmembrane AMPA receptor regulatory proteins

Optical control of NMDA-receptors with a diffusible photoswitch

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity, learning and memory, and are implicated in various neuronal disorders. We synthesized a diffusible photochromic glutamate analogue, azobenzene-triazole-glutamate (ATG), which is specific for NMDARs and functions as a photoswitchable agonist. ATG is inactive in its dark-adapted trans-isoform, but can be converted into its active cis-isoform using one-photon (near UV) or two-photon (740 nm) excitation. Irradiation with violet light photo-inactivates ATG within milliseconds, allowing agonist removal on the timescale of NMDAR deactivation. ATG is compatible with Ca2+ imaging and can be used to optically mimic synaptic coincidence detection protocols. Thus, ATG can be used like traditional caged glutamate compounds, but with the added advantages of NMDAR specificity, low antagonism of GABAR-mediated currents, and precise temporal control of agonist delivery

Auxiliary subunits of voltage-gated Calcium channels as modulators of ionotropic glutamate receptors

Die vorliegende Arbeit untersucht mögliche Wechselwirkungen zwischen AMPA-Rezeptoren und Hilfsuntereinheiten spannungsgesteuerter Calciumkanäle. Es konnte gezeigt werden, dass die Koexpression von alpha-2-delta-Untereinheiten mit AMPA-Rezeptoren in Xenopus laevis-Oozyten die elektrophysiologischen Eigenschaften des Rezeptors verändert. Bei Koexpression in HEK-Zellen hingegen konnte keine solche Modulation festgestellt werden; daraus wurde geschlossen, dass für die Interaktion zwischen alpha-2-delta-Untereinheiten und AMPA-Rezeptoren ein weiteres, bislang unbekanntes Adapterprotein notwendig ist, welches in Xenopus-Oozyten endigen exprimiert wird, nicht jedoch in HEK-Zellen. Diese Hypothese wird weiter unterstützt durch den Umstand, dass durch die Markierung des AMPA-Rezeptors mit einem Fluoreszenzprotein am intrazellulären C-Terminus die Modulation durch die extrazellulär lokalisierten alpha-2-delta-Untereinheiten aufgehoben wurde.This thesis investigates possible interactions between AMPA receptors and auxiliary subunits of voltage-gated calcium channels. It could be shown that coespression of alpha-2-delta subunits with AMPA receptors in Xenopus laevis oocytes altered the electrophysiological properties of the receptor. However, when coexpressed in HEK cells, no such modulation could be detected. It was therefore concluded that the interaction between alpha-2-delta subunits and AMPA receptors requires an additional, thus far unknown adaptor protein, which is endogenously expressed n Xenopus oocytes, but not in HEK cells. This hypothesis is further supported by the fact that a fluorescent protein attached to the C-terminus of the AMPA receptor abolished the modulation by the extracellularly located alpha-2-delta subunit

C-terminal Domains of Transmembrane α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate (AMPA) Receptor Regulatory Proteins Not Only Facilitate Trafficking but Are Major Modulators of AMPA Receptor Function*

α-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 (γ2, γ3, γ4, and γ8) modulate AMPA receptor function remain largely unknown. Previous studies suggested the C-terminal domain (CTD) of γ2 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 γ1, 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 γ4, 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