A truncated Kv1.1 protein in the brain of the megencephaly mouse: expression and interaction

BACKGROUND: The megencephaly mouse, mceph/mceph, is epileptic and displays a dramatically increased brain volume and neuronal count. The responsible mutation was recently revealed to be an eleven base pair deletion, leading to a frame shift, in the gene encoding the potassium channel Kv1.1. The predicted MCEPH protein is truncated at amino acid 230 out of 495. Truncated proteins are usually not expressed since nonsense mRNAs are most often degraded. However, high Kv1.1 mRNA levels in mceph/mceph brain indicated that it escaped this control mechanism. Therefore, we hypothesized that the truncated Kv1.1 would be expressed and dysregulate other Kv1 subunits in the mceph/mceph mice. RESULTS: We found that the MCEPH protein is expressed in the brain of mceph/mceph mice. MCEPH was found to lack mature (Golgi) glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum (ER). Interactions between MCEPH and other Kv1 subunits were studied in cell culture, Xenopus oocytes and the brain. MCEPH can form tetramers with Kv1.1 in cell culture and has a dominant negative effect on Kv1.2 and Kv1.3 currents in oocytes. However, it does not retain Kv1.2 in the ER of neurons. CONCLUSION: The megencephaly mice express a truncated Kv1.1 in the brain, and constitute a unique tool to study Kv1.1 trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth

Role of potassium channels in regulating neuronal activity

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

Functional Characterization of Neurotransmitter Activation and Modulation in a Nematode Model Ligand‐gated Lon Channel

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\u27s 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

Increased carotid artery lesion inflammation upon treatment with the CD137 agonistic antibody 2A

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

A truncated Kv1.1 protein in the brain of the mouse: expression and interaction-0

<p><b>Copyright information:</b></p><p>Taken from "A truncated Kv1.1 protein in the brain of the mouse: expression and interaction"</p><p>BMC Neuroscience 2005;6():65-65.</p><p>Published online 23 Nov 2005</p><p>PMCID:PMC1322225.</p><p>Copyright © 2005 Persson et al; licensee BioMed Central Ltd.</p> SDS-PAGE and immunoblotted with the polyclonal Kv1.1 N-terminal antibody. In wild type lysate a strong band was detected at 86 kDa, corresponding to Kv1.1. The same band was seen in the Kv1.1 null and lysates but at a lower intensity. The 86 kDa bands in Kv1.1 null and lysates are due to antibody cross reactivity since neither Kv1.1 null nor mice have any full-length Kv1.1 protein. B. The polyclonal Kv1.1 N-terminal antibody was preincubated with the peptide used for immunization. Lysate from wild type (+/+) and (m/m) brains were loaded on SDS-PAGE and immunoblotted with the Kv1.1 N-terminal antibody without or after preincubation. The preincubation completely blocked the signal. C. A longer exposure of the immunoblot in panel A. In brain lysate there was a unique band at approximately 30 kDa, which corresponds to the calculated weight of MCEPH (arrow)

A truncated Kv1.1 protein in the brain of the mouse: expression and interaction-2

<p><b>Copyright information:</b></p><p>Taken from "A truncated Kv1.1 protein in the brain of the mouse: expression and interaction"</p><p>BMC Neuroscience 2005;6():65-65.</p><p>Published online 23 Nov 2005</p><p>PMCID:PMC1322225.</p><p>Copyright © 2005 Persson et al; licensee BioMed Central Ltd.</p>ern. Both EndoH and PNGaseF reduced the molecular weight with approximately 3 kDa (arrow). This corresponds to core glycosylation. No unglycosylated MCEPH was detected