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- Heusser, Stephanie A., et al.
(författare)
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Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel
- 2016
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 138:2, s. 243-253
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Tidskriftsartikel (refereegranskat)abstract
- The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluCl(cryst)) as a structurally accessible model for GABA(A) receptors. In contrast to native-like controls, GluCl(cryst) exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABA(A) receptors, and to site-directed mutations predicted to alter channel opening.
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- Klement, Göran
(författare)
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Role of potassium channels in regulating neuronal activity
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The firing behaviour of excitable cells is fundamental for the information processing in multicellular organisms, varying from single spikes to different forms of repetitive firing. Of the many regulators, voltage gated potassium channels play a major role. In this thesis some aspects of the potassium channel regulation of firing are explored. (i) The role of the channel density per se is studied in an in silico model, (ii) the effect of a spontaneously mutated potassium channel is studied in hippocampal slices from a mouse model, (iii) the effect on the expression of potassium channels in general, and consequently on the firing, by this spontaneous mutation is studied in Xenopus oocytes, (iv) the molecular mechanisms giving the hERG channel its specific regulatory role in cardiac firing are studied in Xenopus oocytes and (v) the mechanisms behind the spontaneous current events in hypothalamic neurons, shaping hypothalamic firing patterns, are studied in mechanically isolated cells. The computational study was based on an analysis of a hippocampal interneuron and showed that varying the density of sodium- and potassium channels results in qualitatively different firing patterns and threshold dynamics, mathematically associated with different bifurcation types (saddle node, Hopf and double-orbit). The study of the effects of a mutated potassium channel was performed on a megencephalic mouse model, having a truncated KV1.1 channel gene (mceph). A patch-clamp analysis of neurons in hippocampal slices showed that one effect of the truncation on the neurons was, in addition to an enlarged size, a slight increase in firing frequency, compatible with a decreased density of potassium channels. The study of the MCEPH expression in mceph/mceph mice, showed that it indeed was expressed, but completely retained in the ER. It was also found that it retained other KV1 channels in the ER, reducing their density in the plasma membrane. The study of the molecular mechanism underlying the specific features of hERG was performed by analysing Shaker channels with hERG emulating substitutions. hERG is structurally characterized by aromatic residues in the internal vestibule. We introduced one of these, tyrosine, in Shaker, and found that it induced hERG like features, suggesting that the tyrosine residue has a role in forming the specific hERG kinetics. In addition, the tyrosine substitution induced an inactivation component with inversed voltage-dependence. The study of the spontaneous hypothalamic current events was performed with medial preoptic area neurons and showed that the currents were due to calcium-activated potassium channels of the SK3 subtype, triggered by Ca2+ release from intracellular stores via ryanodine receptor channels. Current clamp measurements showed that the spontaneous current events had a role in shaping the firing patterns of the medial preoptic neurons. In conclusion, this thesis work adds information on the role of potassium channels in regulating neuronal firing at different levels. It suggests ways to understand pharmacological effects on firing patterns, presents a background for future studies on the trafficking of potassium channels, suggests a novel determinant involved in hERG kinetics and indicates a role for SK channels in neuronal firing.
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| 6. |
- Klement, Göran, et al.
(författare)
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Spontaneous ryanodine-receptor-dependent Ca2+-activated K+ currents and hyperpolarizations in rat medial preoptic neurons
- 2010
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Ingår i: Journal of Neurophysiology. - : American Physiological Society. - 0022-3077 .- 1522-1598. ; 103:5, s. 2900-2911
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the present study was to clarify the identity of slow spontaneous currents, the underlying mechanism and possible role for impulse generation in neurons of the rat medial preoptic nucleus (MPN). Acutely dissociated neurons were studied with the perforated patch-clamp technique. Spontaneous outward currents, at a frequency of approximately 0.5 Hz and with a decay time constant of approximately 200 ms, were frequently detected in neurons when voltage-clamped between approximately -70 and -30 mV. The dependence on extracellular K(+) concentration was consistent with K(+) as the main charge carrier. We concluded that the main characteristics were similar to those of spontaneous miniature outward currents (SMOCs), previously reported mainly for muscle fibers and peripheral nerve. From the dependence on voltage and from a pharmacological analysis, we concluded that the currents were carried through small-conductance Ca(2+)-activated (SK) channels, of the SK3 subtype. From experiments with ryanodine, xestospongin C, and caffeine, we concluded that the spontaneous currents were triggered by Ca(2+) release from intracellular stores via ryanodine receptor channels. An apparent voltage dependence was explained by masking of the spontaneous currents as a consequence of steady SK-channel activation at membrane potentials > -30 mV. Under current-clamp conditions, corresponding transient hyperpolarizations occasionally exceeded 10 mV in amplitude and reduced the frequency of spontaneous impulses. In conclusion, MPN neurons display spontaneous hyperpolarizations triggered by Ca(2+) release via ryanodine receptors and SK3-channel activation. Thus such events may affect impulse firing of MPN neurons.
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- Rosén Klement, Maria L., et al.
(författare)
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A processive lipid glycosyltransferase in the small human pathogen Mycoplasma pneumoniae: involvement in host immune response
- 2007
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 65:6, s. 1444-1457
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Tidskriftsartikel (refereegranskat)abstract
- The human pathogen Mycoplasma pneumoniae has a very small genome but with many yet not identified gene functions, e.g. for membrane lipid biosynthesis. Extensive radioactive labelling in vivo and enzyme assays in vitro revealed a substantial capacity for membrane glycolipid biosynthesis, yielding three glycolipids, five phosphoglycolipids, in addition to six phospholipids. Most glycolipids were synthesized in a cell protein/lipid-detergent extract in vitro; galactose was incorporated into all species, whereas glucose only into a few. One (MPN483) of the three predicted glycosyltransferases (GTs; all essential) was both processive and promiscuous, synthesizing most of the identified glycolipids. These enzymes are of a GT-A fold, similar to an established structure, and belong to CAZy GT-family 2. The cloned MPN483 could use both diacylglycerol (DAG) and human ceramide acceptor substrates, and in particular UDP-galactose but also UDP-glucose as donors, making mono-, di- and trihexose variants. MPN483 output and processitivity was strongly influenced by the local lipid environment of anionic lipids. The structure of a major β1,6GlcβGalDAG species was determined by NMR spectroscopy. This, as well as other purified M. pneumoniae glycolipid species, is important antigens in early infections, as revealed from ELISA screens with patient IgM sera, highlighting new aspects of glycolipid function.
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| 9. |
- Söderström, Leif, et al.
(författare)
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Increased carotid artery lesion inflammation upon treatment with the CD137 agonistic antibody 2A
- 2017
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Ingår i: Circulation Journal. - 1346-9843. ; 81:12, s. 1945-1952
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Tidskriftsartikel (refereegranskat)abstract
- Background: Increased inflammatory activity destabilizes the atherosclerotic lesion and may lead to atherothrombosis and symptomatic cardiovascular disease. Co-stimulatory molecules, such as CD137, are key regulators of inflammation, and CD137 activity regulates inflammation in experimental atherosclerosis. Here, we hypothesized that CD137 activation promotes carotid artery inflammation and atherothrombosis. Methods and Results: In a model of inducible atherothrombosis with surgical ligation of the right carotid artery and a subsequent placement of a polyethene cuff, elevated levels of CD137 and CD137 ligand mRNA in atherothrombotic vs. non-atherothrombotic murine carotid lesions was observed. Mice treated with the CD137 agonistic antibody 2A showed signs of increased inflammation in the aorta and a higher proportion of CD8+ T cells in spleen and blood. In carotid lesions of 2A-treated mice, significantly higher counts of CD8+ and major histocompatibility (MHC)-class II molecule I-Ab+ cells were observed. Treatment with the CD137 agonistic antibody 2A did not significantly affect the atherothrombosis frequency in 16-week-old mice in this model. Conclusions: Levels of CD137 and CD137 ligand mRNA were higher in advanced atherosclerotic disease compared to control vessels, and treatment with the CD137 agonistic antibody 2A, in a murine model for inducible atherothrombosis promoted vascular inflammation, but had no significant effect on atherothrombosis frequency at this early disease stage.
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