Ligand-binding of Cys-loop receptors results in rearrangements of extracellular loop structures which are further translated into the tilting of membrane spanning helices, and finally opening of the ion channels. The cryo-EM structure of the homopentameric α1 glycine receptor (GlyR) demonstrated an involvement of the extracellular β8-β9 loop in the transition from ligand-bound receptors to the open channel state. Recently, we identified a functional role of the β8-β9 loop in a novel startle disease mouse model shaky. The mutation of residue GlyRα1Q177 to lysine present in shaky mice resulted in reduced glycine potency, reduced synaptic expression, and a disrupted hydrogen network at the structural level around position GlyRα1Q177. Here, we investigated the role of amino acid volume, side chain length, and charge at position Q177 to get deeper insights into the functional role of the β8-β9 loop. We used a combined approach of in vitro expression analysis, functional electrophysiological recordings, and GlyR modeling to describe the role of Q177 for GlyR ion channel function. GlyRα1Q177 variants do not disturb ion channel transport to the cellular surface of transfected cells, neither in homomeric nor in heteromeric GlyR configurations. The EC50 values were increased for all GlyRα1Q177 variants in comparison to the wild type. The largest decrease in glycine potency was observed for the variant GlyRα1Q177R. Potencies of the partial agonists β-alanine and taurine were also reduced. Our data are further supported by homology modeling. The GlyRα1Q177R variant does not form hydrogen bonds with the surrounding network of residue Q177 similar to the substitution with a basic lysine present in the mouse mutant shaky. Among all investigated Q177 mutants, the neutral exchange of glutamine to asparagine as well as the introduction of the closely related amino acid glutamic acid preserve the hydrogen bond network. Introduction of amino acids with small side chains or larger volume resulted in a loss of their hydrogen bonds to neighboring residues. The β8-β9 loop is thus an important structural and functional determinant of the inhibitory GlyR.
Abstract Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABA A receptors (GABA A Rs). In this study, the modulatory potential of 11 SQTs at GABA A Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole‐cell recordings. Significantly different effects of β‐caryophyllene and α‐humulene, as well as their respective derivatives β‐caryolanol and humulol, were observed in the HEK293 cell system. In neurons, the concomitant presence of phasic and tonic GABA A R configurations accounts for differences in receptor modulation by SQTs. The in vivo presence of the γ 2 and δ subunits is important for SQT modulation. While phasic GABA A receptors in hippocampal neurons exhibited significantly altered GABA‐evoked current amplitudes in the presence of humulol and guaiol, negative allosteric potential at recombinantly expressed α 1 β 2 γ 2 receptors was only verified for humolol. Modeling and docking studies provided support for the binding of SQTs to the neurosteroid‐binding site of the GABA A R localized between transmembrane segments 1 and 3 at the ( + α)‐( ‐ α) interface. In sum, differences in the modulation of GABA A R isoforms between SQTs were identified. Another finding is that our results provide an indication that nutritional digestion affects the neurotropic potential of natural compounds. image
Abstract Background: Glioblastoma multiforme (GBM) and triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with very poor patient outcomes. GBM uses axons and vessels as migratory cues to disseminate, however it is not known, if TNBC shares the same behavior. There is a need to understand brain tumor cell spreading and if GBM and TNBC have similar migration properties involving the signaling pathway RHOB-ROCK-PTEN. We tested for durotaxis, adherence and migration of GBM and TNBC using live-cell imaging and performed molecular analyses on three-dimensional (3D) structures. Methods: Aligned 3D printed scaffolds and microfibers were designed to mimic brain axon tracts and vessels for migration. GBM and TNBC cell lines, each with opposing PTEN genotypes, were analysed with RHO, ROCK and PTEN inhibitors and rescuing PTEN function using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while SEM, confocal microscopy and cell tracking addressed cell morphology. Results: GBM and TNBC with homozygote PTEN loss of function and RHOB high expression were amoeboid shaped and demonstrated enhanced durotaxis, adhesion and migration on 3D microfibers, in contrast to PTEN wildtype GBM and TNBC showing elongated cells and low RHOB. RNA-sequencing exhibited that RHOB was significantly the highest expressed gene in GBM PTEN loss of function cells. Pathway inhibitors and PTEN rescue of function verified an essential role of RHOB-ROCK-PTEN signaling for durotaxis, adhesion, migration, cell morphology and plasticity using 3D printed microfibers. Conclusions: This study validates a significant role of a PTEN genotype for cellular properties including durotaxis, adhesion and migration. GBM and TNBC cells with PTEN loss of function have a greater affinity for stiffer brain structures promoting metastasis. We propose the significance of PTEN and RHOB in cellular oncology not only for primary tumors, but also for metastasizing tumors, where RHOB inhibitors could play an essential role for improved therapy.
Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures.3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology.In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes.This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.
Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric α1 and heteromeric α1-β GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 μM and 0.5 μM. EC50 of glycine increased upon application of the protein kinase activators Forskolin and phorbol-12-myristate-13-acetate (PMA) but decreased in the presence of the PKC inhibitor Staurosporine aglycon and the PKA inhibitor H-89. Desensitization of recombinant α1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric α1-β GlyRs. The time course of receptor activation was determined for homomeric α1 receptors and revealed two simultaneous effects: cells showed a decrease of EC50 after 3-6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC50, which overlay and eventually exceeded the cells intrinsic variation of EC50. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA- and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.
Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.
Abstract Impairments in neuronal circuits underly multiple neurodevelopmental and neurodegenerative disorders. 3D cell culture models enhance the complexity of in vitro systems and provide a microenvironment closer to the native situation than with 2D cultures. Such novel model systems will allow the assessment of neuronal network formation and their dysfunction under disease conditions. Here, mouse cortical neurons are cultured from embryonic day E17 within in a fiber‐reinforced matrix. A soft Matrigel with a shear modulus of 31 ± 5.6 Pa is reinforced with scaffolds created by melt electrowriting, improving its mechanical properties and facilitating the handling. Cortical neurons display enhance cell viability and the neuronal network maturation in 3D, estimated by staining of dendrites and synapses over 21 days in vitro, is faster in 3D compared to 2D cultures. Using functional readouts with electrophysiological recordings, different firing patterns of action potentials are observed, which are absent in the presence of the sodium channel blocker, tetrodotoxin. Voltage‐gated sodium currents display a current–voltage relationship with a maximum peak current at −25 mV. With its high customizability in terms of scaffold reinforcement and soft matrix formulation, this approach represents a new tool to study neuronal networks in 3D under normal and, potentially, disease conditions.
Abstract 2D electrophysiology is often used to determine the electrical properties of neurons. In the brain however, neurons form extensive 3D networks. Thus, performing electrophysiology in a 3D environment provides a closer situation to the physiological condition and serves as a useful tool for various applications in the field of neuroscience. In this study, 3D electrophysiology is established within a fiber‐reinforced matrix to enable fast readouts from transfected cells, which are often used as model systems for 2D electrophysiology. Using melt electrowriting (MEW) of scaffolds to reinforce Matrigel, 3D electrophysiology is performed on a glycine receptor‐transfected Ltk‐11 mouse fibroblast cell line. The glycine receptor is an inhibitory ion channel associated when mutated with impaired neuromotor behavior. The average thickness of the MEW scaffold is 141.4 ± 5.7 µm, using 9.7 ± 0.2 µm diameter fibers, and square pore spacings of 100, 200, and 400 µm. For the first time, the electrophysiological characterization of glycine receptor‐transfected cells is demonstrated with respect to agonist efficacy and potency in a 3D matrix. With the MEW scaffold reinforcement not interfering with the electrophysiological measurement, this approach can now be further adapted and developed for different kinds of neuronal cultures to study and understand pathological mechanisms under disease conditions.
