Protein phosphatase 1 modulates the inhibitory effect of With-no-Lysine kinase 4 on ROMK channels

With-no-Lysine kinase 4 (WNK4) inhibited ROMK (Kir1.1) channels and the inhibitory effect of WNK4 was abolished by serum-glucocorticoid-induced kinase 1 (SGK1) but restored by c-Src. The aim of the present study is to explore the mechanism by which Src-family tyrosine kinase (SFK) modulates the effect of SGK1 on WNK4 and to test the role of SFK-WNK4-SGK1 interaction in regulating ROMK channels in the kidney. Immunoprecipitation demonstrated that protein phosphatase 1 (PP1) binds to WNK4 at amino acid (aa) residues 695-699 (PP1(#1)) and at aa 1211-1215 (PP1(#2)). WNK4(-PP1#1) and WNK4(-PP1#2), in which the PP1(#1) or PP1(#2) binding site was deleted or mutated, inhibited ROMK channels as potently as WNK4. However, c-Src restored the inhibitory effect of WNK4 but not WNK4(-PP1#1) on ROMK channels in the presence of SGK1. Moreover, expression of c-Src inhibited SGK1-induced phosphorylation of WNK4 but not WNK4(-PP1#1) at serine residue 1196 (Ser(1196)). In contrast, coexpression of c-Src restored the inhibitory effect of WNK4(-PP1#2) on ROMK in the presence of SGK1 and diminished SGK1-induced WNK4 phosphorylation at Ser(1196) in cells transfected with WNK4(-PP1#2). This suggests the possibility that c-Src regulates the interaction between WNK4 and SGK1 through activating PP1 binding to aa 695-9 thereby decreasing WNK4 phosphorylation and restoring the inhibitory effect of WNK4. This mechanism plays a role in suppressing ROMK channel activity during the volume depletion because inhibition of SFK or serine/threonine phosphatases increases ROMK channel activity in the cortical collecting duct of rats on a low-Na diet. We conclude that regulation of phosphatase activity by SFK plays a role in determining the effect of aldosterone on ROMK channels and on renal K secretion.     

Resolution of sertraline with (R)-mandelic acid: chiral discrimination mechanism study

The chiral discrimination mechanism in the resolution of sertraline with mandelic acid was investigated by examining the weak intermolecular interactions (such as hydrogen bond, CH/π, and van der Waals interactions) and molecular packing difference in crystal structures of the resulting diastereomeric salts. A new one-dimensional chain-like hydrogen-bonding network and unique supramolecular packing mode are disclosed. The investigation demonstrated that stable hydrogen-bonding pattern, herringbone-like arrangement of aromatic rings, and planar boundary surface in the hydrophobic region are the three most important structural characteristics expected in less soluble diastereomeric salts. The existence and magnitude of hydrogen bond, CH/π interaction, and van der Waals interaction related to three characteristic structures, determine the stability of diastereomeric salt. The hydrogen bond is not necessarily the dominant factor while the synergy and optimization of all weak intermolecular interactions attribute to the chiral recognition.     

AMP-activated protein kinase β-subunit requires internal motion for optimal carbohydrate binding

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β(1) and β(2)). Muscle-specific β(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β(2)-CBM is concurrent with an increase in flexibility in β(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to β(1)-CBM, unbound β(2)-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β(2)-CBM mutant. Insertion of a threonine into the background of β(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.     

Identification of Contact Sites between Ankyrin and Band 3 in the Human Erythrocyte Membrane

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.     

Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion

Adult-born granule cells (GCs), a minor population of cells in the hippocampal dentate gyrus, are highly active during the first few weeks after functional integration into the neuronal network, distinguishing them from less active, older adult-born GCs and the major population of dentate GCs generated developmentally. To ascertain whether young and old GCs perform distinct memory functions, we created a transgenic mouse in which output of old GCs was specifically inhibited while leaving a substantial portion of young GCs intact. These mice exhibited enhanced or normal pattern separation between similar contexts, which was reduced following ablation of young GCs. Furthermore, these mutant mice exhibited deficits in rapid pattern completion. Therefore, pattern separation requires adult-born young GCs but not old GCs, and older GCs contribute to the rapid recall by pattern completion. Our data suggest that as adult-born GCs age, their function switches from pattern separation to rapid pattern completion.     

Road mortality in freshwater turtles: identifying causes of spatial patterns to optimize road planning and mitigation

Road mortality of freshwater turtles can be high enough to imperil populations near roads, thus there is a need to efficiently and accurately locate regions of excessive road-kill along road networks for mitigation. Weekly over 2?years, we drove a 160?km highway circuit in northeastern New York State, USA and recorded the location of all detected road-kill of three freshwater turtle species (Chelydra serpentina, Chrysemys picta, Emydoidea blandingii). We then analyzed the spatial dispersion of road-kill and the road and landscape features associated with road-kill locations. Road-kill was most prevalent at a limited number of short road segments, termed ‘hotspots’. The locations of hotspots, as indicated by kernel density analysis, and the peak spatial extent of hotspots (250?m), as indicated by Ripley’s?K, corresponded to the locations and average lengths of causeways (road segments with wetlands within 100?m on both sides). Hotspots were located at causeways that were greater than 200?m length and characterized by high traffic volumes, close proximity to water, and high forest coverage. We conclude that freshwater turtle road mortality is spatially aggregated at short, severe hotspots, and hotspot locations can be predicted when the locations of wetlands, traffic volumes, and the land-uses bordering roads are known. Hotspot models using these predictors can locate sites along a road network that are the most promising for mitigation to reduce excessive road mortality and maintain connectivity.     

The abundance of Ixodes ricinus ticks depends on tree species composition and shrub cover

SUMMARY The mainstream forestry policy in many European countries is to convert coniferous plantations into (semi-natural) deciduous woodlands. However, woodlands are the main habitat for Ixodes ricinus ticks. Therefore, assessing to what extent tick abundance and infection with Borrelia spirochetes are affected by forest composition and structure is a prerequisite for effective prevention of Lyme borreliosis. We selected a total of 25 pine and oak stands, both with and without an abundant shrub layer, in northern Belgium and estimated tick abundance between April and October 2008-2010. Additionally, the presence of deer beds was used as an indicator of relative deer habitat use. Borrelia infections in questing nymphs were determined by polymerase chain reactions. The abundance of larvae, nymphs, and adults was higher in oak stands compared to pine stands and increased with increasing shrub cover, most likely due to differences in habitat use by the ticks' main hosts. Whereas tick abundance was markedly higher in structure-rich oak stands compared to homogeneous pine stands, the Borrelia infection rates in nymphs did not differ significantly. Our results indicate that conversion towards structure-rich deciduous forests might create more suitable tick habitats, but we were unable to detect an effect on the infection rate.