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 AMPA receptors using a photoswitchable quinoxaline-2,3-dione antagonist

AMPA receptors respond to the neurotransmitter glutamate and play a critical role in excitatory neurotransmission. They have been implicated in several psychiatric disorders and have rich pharmacology. Antagonists of AMPA receptors have been explored as drugs and one has even reached the clinic. We now introduce a freely diffusible photoswitchable antagonist that is selective for AMPA receptors and endows them with light-sensitivity. Our photoswitch, ShuBQX-3, is active in its dark-adapted trans-isoform but is significantly less active as its cis-isoform. ShuBQX-3 exhibits a remarkable red-shifting of its photoswitching properties through interactions with the AMPA receptor ligand binding site. Since it can be used to control action potential firing with light, it could emerge as a powerful tool for studying synaptic transmission with high spatial and temporal precision

Distal chromatin structure influences local nucleosome positions and gene expression

The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called ‘antinucleosomal elements’ like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression

Modulation of GluN3B-containing excitatory glycine receptors by zinc

Im zentralen Nervensystem spielt die Klasse der N-Methyl-D-aspartat (NMDA)-Rezeptoren eine entscheidende Rolle für Lernmechanismen und das Gedächtnis. Neben der Glycin-bindenden GluN1- und der Glutamat-bindenden GluN2-Untereinheit existiert die Glycin-bindende GluN3-Untereinheit. Ein Rezeptor bestehend aus GluN1 und GluN3 wird durch die Bindung von Glycin alleine aktiviert. Zunächst wurde das Verhalten GluN1/GluN3B-haltiger Rezeptoren gegenüber Zink und einem Antagonisten der Glycinbindestelle der GluN1-Untereinheit, MDL-29951, untersucht und mit GluN1/GluN2-haltigen Rezeptoren verglichen. Zudem wurden die beteiligten Sequenzelemente des Rezeptors untersucht. Des Weiteren wurde das Verhalten GluN1/GluN3A- und GluN1/GluN3B-haltiger Rezeptoren gegenüber Zink und MDL-29951 verglichen. Zusätzlich wurde das Verhalten GluN1-a/GluN3A- und GluN1-b/GluN3A-haltiger Rezeptoren gegenüber Protonen getestet. Zuletzt wurde der Plasmamembranexport der GluN1-3-Spleißvariante untersucht.In the central nervous system, the N-methyl-D-asparte (NMDA) receptors play a crucial role for learning and memory. Besides the glycine-binding GluN1 and the glutamate-binding GluN2 subunits, the glycine-binding GluN3 subunit was found. This subunit, when coexpressed with GluN1, leads to a receptor that is activated by glycine alone. First, the behavior of GluN1/GluN3B-containing receptors towards zinc and the glycine binding site antagonist MDL-29951 was compared to the behavior of conventional, GluN1/GluN2-containing receptors. Also, a closer look was taken into the specific sequence elements that mediate these effects. Additionally, GluN1/GluN3A- and GluN1/GluN3B-containing receptors were compared with regard to their modulation by zinc and MDL-29951. Furthermore, the proton sensitivity of GluN1-a/GluN3A and GluN1-b/GluN3A-containing receptors was investigated. Finally, a look into the plasma membrane export of the GluN1-3 splice variant of the GluN1 subunit was taken

Optical control of AMPA receptors using a photoswitchable quinoxaline-2,3-dione antagonist

AMPA receptors respond to the neurotransmitter glutamate and play a critical role in excitatory neurotransmission. They have been implicated in several psychiatric disorders and have rich pharmacology. Antagonists of AMPA receptors have been explored as drugs and one has even reached the clinic. We now introduce a freely diffusible photoswitchable antagonist that is selective for AMPA receptors and endows them with light-sensitivity. Our photoswitch, ShuBQX-3, is active in its dark-adapted trans-isoform but is significantly less active as its cis-isoform. ShuBQX-3 exhibits a remarkable red-shifting of its photoswitching properties through interactions with the AMPA receptor ligand binding site. Since it can be used to control action potential firing with light, it could emerge as a powerful tool for studying synaptic transmission with high spatial and temporal precision

The Influence of Breast Tumour-Derived Factors and Wnt Antagonism on the Transformation of Adipose-Derived Mesenchymal Stem Cells into Tumour-Associated Fibroblasts

Within the tumour stroma, a heterogeneous population of cell types reciprocally regulates cell proliferation, which considerably affects the progression of the disease. In this study, using tumour conditioned medium (TCM) derived from breast tumour cell lines – MCF7 and MDA MB 231, we have demonstrated the differentiation of adipose-derived mesenchymal stem cells (ADSCs) into tumour-associated fibroblasts (TAFs). Since the Wnt signalling pathway is a key signalling pathway driving breast tumour growth, the effect of the Wnt antagonist secreted frizzled-related protein 4 (sFRP4) was also examined. The response of ADSCs to TCM and sFRP4 treatments was determined by using cell viability assay to determine the changes in ADSC viability, immunofluorescence for mesenchymal markers, glucose uptake assay, and glycolysis stress test using the Seahorse Extracellular Flux analyser to determine the glycolytic activity of ADSCs. ADSCs have been shown to acquire a hyper-proliferative state, significantly increasing their number upon short-term and long-term exposure to TCM. Changes have also been observed in the expression of key mesenchymal markers as well as in the metabolic state of ADSCs. SFRP4 significantly inhibited the differentiation of ADSCs into TAFs by reducing cell growth as well as mesenchymal marker expression (cell line-dependent). However, sFRP4 did not induce further significant changes to the altered metabolic phenotype of ADSCs following TCM exposure. Altogether, this study suggests that the breast tumour milieu may transform ADSCs into a tumour-supportive phenotype, which can be altered by Wnt antagonism, but is independent of metabolic changes

The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences