The inhibitory glycine receptor (GlyR) mediates synaptic inhibition in the spinal cord, brain stem, and other regions of the mammalian central nervous system. Glucose was shown to potentiate α1 GlyRs by interacting with K143. Here, additional amino acids involved in glucose modulation were identified using a structure-based approach of site-directed mutagenesis followed by whole-cell patch-clamp analysis. We identified two additional lysine residues in the α1 GlyR extracellular domain, K16 and K281, that were involved in glucose modulation. Mutation of either residue to alanine abolished glucose potentiation. Residue K281 is located in the same pocket as K143 and could thus contribute to glucose binding. The double mutant K143A-K281A showed a 6-fold increase of EC50, while EC50 of both single mutants K143A and K281A was only slightly increased (1.7- and 1.3-fold, respectively). K16 is located at an analgesic binding site that is distant from the agonist or glucose sites, and the K16A mutation may generate a receptor species that is not potentiated. GlyR position α1-S267 is close to the postulated glucose binding site and known for interactions with ethanol and anesthetics. In the presence of glucose, GlyR α1 mutants S267A, S267I, and S267R showed potentiation, no effect, and reduction of current responses, respectively. This pattern follows that of ethanol modulation and suggests that the interaction sites of glucose and ethanol are identical or located close to each other. Our results support the presence of a distinct binding site for glucose on the glycine receptor, overlapping with the ivermectin/ethanol binding pocket near the transmembrane region and the TM2-3 loop.

Alanine

10.1021/acschemneuro.0c00566

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Kavalactones from Kava (Piper methysticum) root extract as modulators of recombinant human glycine receptors

Biological Chemistry (2019)

Nada Hany Hegazy Hans‐Georg Breitinger Ulrike Breitinger

Abstract Roots of kava ( Piper methysticum ) plant are used in almost all Pacific Ocean cultures to prepare a drink with sedative, anesthetic and euphoric properties. One of the main active ingredients of the extract are kava lactones. Here, kava root CO 2 extract and three kavalactones, DL-kavain, dihydrokavain and yangonin (isolated from whole extract by column chromatography) were tested for their inhibitory action on recombinant homomeric human α1 glycine receptors expressed in HEK293 cells. Kava CO 2 root extract, as well as the individual components DL-kavain, dihydrokavain and yangonin inhibited glycine receptor activity in a dose-dependent manner. DL-kavain was the most potent inhibitor (IC 50 = 0.077 ± 0.002 m m ), followed by yangonin (IC 50 = 0.31 ± 0.04 m m ) and dihydrokavain (IC 50 = 3.23 ± 0.10 m m ) which were 4- and 40-fold less active than DL-kavain, respectively. Application of kava root extract did not reduce maximum currents, but increased EC 50 of glycine. Simultaneous application of kava extract and strychnine showed additive inhibition, suggesting that binding of kavalactones and strychnine on the receptor is mutually exclusive. Overall, kavalactones exert a moderate inhibitory effect on the human α1 glycine receptor with DL-kavain being the most potent constituent.

Kava

10.1515/hsz-2019-0112

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Liposomal delivery of functional transmembrane ion channels into the cell membranes of target cells; a potential approach for the treatment of channelopathies

International Journal of Biological Macromolecules (2019)

Shahenda Ramadan Salma N. Tammam Maryam A. Shetab Boushehri Hans‐Georg Breitinger Ulrike Breitinger

HEK 293 cells

Cell membrane

Transmembrane channels

10.1016/j.ijbiomac.2019.10.238

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X-Ray Structure of 12-Oxophytodienoate Reductase 1 Provides Structural Insight into Substrate Binding and Specificity within the Family of OYE

Structure (2001)

Constanze Breithaupt Jochen Strassner Ulrike Breitinger R. Huber Peter Macheroux

10.1016/s0969-2126(01)00602-5

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Citations (116)

PKA and PKC Modulators Affect Ion Channel Function and Internalization of Recombinant Alpha1 and Alpha1-Beta Glycine Receptors

Frontiers in Molecular Neuroscience (2018)

Ulrike Breitinger Lamia'a Bahnassawy Dieter Janzen Vera Roemer Cord‐Michael Becker

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.

Homomeric

Staurosporine

10.3389/fnmol.2018.00154

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Glucose is a positive modulator for the activation of human recombinant glycine receptors

Journal of Neurochemistry (2015)

Ulrike Breitinger Karim Raafat Hans‐Georg Breitinger

The inhibitory glycine receptor (GlyR), a cys-loop ion channel receptor, mediates rapid synaptic inhibition in spinal cord, brainstem and higher centres of the mammalian central nervous system. Here, modulation of GlyR function by glucose and fructose was examined in recombinant alpha1 and alpha1/beta GlyRs using patch-clamp methods. Glucose was a positive modulator of the receptor, reducing the average EC50 for glycine up to 4.5-fold. Glucose reduced cell-to-cell variability of glycine-mediated currents by stabilizing receptors with low EC50. Pre-incubation with sugars for several hours also produced augmentation of current responses that persisted after sugar removal. Potentiation by sugars was most significant in the range between 5 and 20 mM, with EC50 values ~ 10 mM, i.e. at physiological levels. Addition of glucose had no significant influence on responses mediated by the other GlyR agonists like taurine, β-alanine or ivermectin, indicating that glucose specifically augmented glycine receptor-mediated responses, and did not act through indirect metabolic effects. Receptor modulation by glucose may account for differences in constants reported in the literature and may be clinically relevant for disorders with elevated blood glucose levels. Glucose and related sugars are essential metabolites. We identified glucose and fructose as positive modulators of the human inhibitory glycine receptor, a neuronal ligand-gated ion channel. Receptor-mediated currents were enhanced at physiological concentrations (~ 10 mM of sugar). Direct modulation of a synaptic receptor by glucose is relevant in clinical cases of elevated blood glucose, and may be considered in experimental protocols.

10.1111/jnc.13215

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Citations (19)