Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P2. In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P2 in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory

In Vitro Analyses of Novel HCN4 Gene Mutations

Background/Aims: The hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 contributes significantly to the generation of basic cardiac electrical activity in the sinus node and is a mediator of modulation by β–adrenergic stimulation. Heterologous expression of sick sinus syndrome (SSS) and bradycardia associated mutations within the human HCN4 gene results in altered channel function. The main aim was to describe the functional characterization of three (two novel and one known) missense mutations of HCN4 identified in families with SSS. Methods: Here, the two-electrode voltage clamp technique on Xenopus laevis oocytes and confocal imaging on transfected COS7 cells respectively, were used to analyze the functional effects of three HCN4 mutations; R378C, R550H, and E1193Q. Membrane surface expressions of wild type and the mutant channels were assessed by confocal microscopy, chemiluminescence assay, and Western blot in COS7 and HeLa cells. Results: The homomeric mutant channels R550H and E1193Q showed loss of function through increased rates of deactivation and distinctly reduced surface expression in all three homomeric mutant channels. HCN4 channels containing R550H and E1193Q mutant subunits only showed minor effects on the voltage dependence and rates of activation/deactivation. In contrast, homomeric R378C exerted a left-shifted activation curve and slowed activation kinetics. These effects were reduced in heteromeric co-expression of R378C with wild-type (WT) channels. Conclusion: Dysfunction of homomeric/heteromeric mutant HCN4-R378C, R550H, and E1193Q channels in the present study was primarily caused by loss of function due to decreased channel surface expression

Discovery of a Novel Activator of KCNQ1-KCNE1 K+ Channel Complexes

KCNQ1 voltage-gated K+ channels (Kv7.1) associate with the family of five KCNE peptides to form complexes with diverse gating properties and pharmacological sensitivities. The varied gating properties of the different KCNQ1-KCNE complexes enables the same K+ channel to function in both excitable and non excitable tissues. Small molecule activators would be valuable tools for dissecting the gating mechanisms of KCNQ1-KCNE complexes; however, there are very few known activators of KCNQ1 channels and most are ineffective on the physiologically relevant KCNQ1-KCNE complexes. Here we show that a simple boronic acid, phenylboronic acid (PBA), activates KCNQ1/KCNE1 complexes co-expressed in Xenopus oocytes at millimolar concentrations. PBA shifts the voltage sensitivity of KCNQ1 channel complexes to favor the open state at negative potentials. Analysis of different-sized charge carriers revealed that PBA also targets the permeation pathway of KCNQ1 channels. Activation by the boronic acid moiety has some specificity for the Kv7 family members (KCNQ1, KCNQ2/3, and KCNQ4) since PBA does not activate Shaker or hERG channels. Furthermore, the commercial availability of numerous PBA derivatives provides a large class of compounds to investigate the gating mechanisms of KCNQ1-KCNE complexes

What's in a name?: a discussion paper on the labels and location of self-organising community groups, with particular reference to mental health and Black groups

This article discusses the labelling and location of self-organising community groups – ‘self-help’, ‘peer support’ and ‘service user’. It notes the increasingly close relationship between these groups and statutory authorities, and how this relationship may put the benefits of the groups at risk. Historical, cultural and social factors are discussed to help explain differences and separate developments within African, Caribbean and other Black communities

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K+ channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the “helix-bundle crossing”. However, in the inwardly rectifying (Kir) potassium channel family, the role of this “hinge” residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper “hinge” residues are in close contact with the base of the pore α-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the “lower” gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue

Wintering grounds, population size and evolutionary history of a cryptic passerine species from isotopic and genetic data

Cryptic species pose a particular challenge to biologists in the context of life history investigations because of the difficulty in their field discrimination. Additionally, there is normally a lag in their widespread acceptance by the scientific community once they are formally recognised. These two factors might constrain our ability to properly assess the conservation status of the different species conforming a cryptic complex. In this study, we analysed isotopic and genetic data to shed light into the still unclear wintering grounds, population size and evolutionary history of the Iberian chiffchaff Phylloscopus ibericus, a species included within the common chiffchaff Phylloscopus collybita until two decades ago due to their phenotypic similarity. We used molecular methods to identify spring-migrating Phylloscopus species captured in northern Iberia, and by comparing the Hydrogen isotopic ratios of their claw tips (δ2Hc; which would reflect the signatures of their wintering grounds), we detected that δ2Hc values of Iberian chiffchaffs were similar to willow warblers (Phylloscopus trochilus; a renowned trans-Saharan migrant), and higher than common chiffchaffs (mostly a pre-Saharan migrant). These results strongly support the idea that Iberian chiffchaffs winter in tropical Africa. We additionally reconstructed the phylogeny and evolutionary history of the Iberian chiffchaff's clade using mitochondrial and nuclear markers. Our results revealed relatively high values of nucleotide diversity (and, hence, high Ne) for the species that were greater than the values of the common/Iberian most recent common ancestor. This suggests that the Iberian chiffchaff did not experience strong bottlenecks after diverging from the common chiffchaff approximately one million years ago. Ultimately, our study provides another illustrative example of how isotopic and genetic analysis tools can help to enhance our understanding of avian ecology and evolution.Depto. de Biodiversidad, Ecología y EvoluciónFac. de Ciencias BiológicasTRUEpu

Regulation of Kainate Receptor Subunit mRNA by Stress and Corticosteroids in the Rat Hippocampus

Kainate receptors are a class of ionotropic glutamate receptors that have a role in the modulation of glutamate release and synaptic plasticity in the hippocampal formation. Previous studies have implicated corticosteroids in the regulation of these receptors and recent clinical work has shown that polymorphisms in kainate receptor subunit genes are associated with susceptibility to major depression and response to anti-depressant treatment. In the present study we sought to examine the effects of chronic stress and corticosteroid treatments upon the expression of the mRNA of kainate receptor subunits GluR5-7 and KA1-2. Our results show that, after 7 days, adrenalectomy results in increased expression of hippocampal KA1, GluR6 and GluR7 mRNAs, an effect which is reversed by treatment with corticosterone in the case of KA1 and GluR7 and by aldosterone treatment in the case of GluR6. 21 days of chronic restraint stress (CRS) elevated the expression of the KA1 subunit, but had no effect on the expression of the other subunits. Similarly, 21 days of treatment with a moderate dose of corticosterone also increased KA1 mRNA in the dentate gyrus, whereas a high corticosterone dose has no effect. Our results suggest an interaction between hippocampal kainate receptor composition and the hypothalamic-pituitary-adrenal (HPA) axis and show a selective chronic stress induced modulation of the KA1 subunit in the dentate gyrus and CA3 that has implications for stress-induced adaptive structural plasticity

K(2P)18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K(2P)18.1 is a relevant regulator. Here, we identify K(2P)18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-kappa B-mediated K(2P)18.1 upregulation in tTreg progenitors. K(2P)18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-kappa B- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K(2P)18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K(2P)18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K(2P)18.1 variant that is associated with poor clinical outcomes indicate that K(2P)18.1 also plays a role in human Treg development. Pharmacological modulation of K(2P)18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K(2P)18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K(2P)18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.Peer reviewe

Axial and Radial Forces of Cross-Bridges Depend on Lattice Spacing

Nearly all mechanochemical models of the cross-bridge treat myosin as a simple linear spring arranged parallel to the contractile filaments. These single-spring models cannot account for the radial force that muscle generates (orthogonal to the long axis of the myofilaments) or the effects of changes in filament lattice spacing. We describe a more complex myosin cross-bridge model that uses multiple springs to replicate myosin's force-generating power stroke and account for the effects of lattice spacing and radial force. The four springs which comprise this model (the 4sXB) correspond to the mechanically relevant portions of myosin's structure. As occurs in vivo, the 4sXB's state-transition kinetics and force-production dynamics vary with lattice spacing. Additionally, we describe a simpler two-spring cross-bridge (2sXB) model which produces results similar to those of the 4sXB model. Unlike the 4sXB model, the 2sXB model requires no iterative techniques, making it more computationally efficient. The rate at which both multi-spring cross-bridges bind and generate force decreases as lattice spacing grows. The axial force generated by each cross-bridge as it undergoes a power stroke increases as lattice spacing grows. The radial force that a cross-bridge produces as it undergoes a power stroke varies from expansive to compressive as lattice spacing increases. Importantly, these results mirror those for intact, contracting muscle force production