Involvement of potassium channels in the antidepressant-like effect of venlafaxine in mice

AimsStudies have shown that the acute administration of venlafaxine elicits an antidepressant-like effect in the mouse forced swim test (FST) by a mechanism dependent on the l-arginine–nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway. Because it has been reported that NO activates different types of potassium (K⁺) channels in the brain, this study investigated the involvement of K⁺ channels in the antidepressant-like effect of venlafaxine in the mouse FST.Main methodsMale adult Swiss mice were pretreated with different K⁺ channel inhibitors or openers 15 min before venlafaxine administration. After 30 min, the open-field test (OFT) and FST were carried out.Key findingsIntracerebroventricular (i.c.v.) pretreatment of mice with subeffective doses of tetraethylammonium (TEA, a non-specific inhibitor of K⁺ channels, 25 pg/site), glibenclamide (an ATP-sensitive K⁺ channel inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K⁺ channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K⁺ channel inhibitor, 10 pg/site) was able to potentiate the action of a subeffective dose of venlafaxine (2 mg/kg, i.p.). Moreover, the reduction in the immobility time elicited by an effective dose of venlafaxine (8 mg/kg, i.p.) in the FST was prevented by the pretreatment of mice with the K⁺ channel openers cromakalim (10 µg/site, i.c.v.) and minoxidil (10 µg/site, i.c.v.). The drugs used in this study did not produce any change in locomotor activity.SignificanceThe results demonstrate that the neuromodulatory effects of venlafaxine, via the inhibition of K⁺ channels, are possibly involved in its anti-immobility activity in the mouse FST.     

YPED:An Integrated Bioinformatics Suite and Database for Mass Spectrometry-based Proteomics Research

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database(YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a singlelaboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry(LC–MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring(MRM)/selective reaction monitoring(SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results.     

Acoustic telemetry reveals cryptic residency of whale sharks

Although whale sharks (Rhincodon typus) have been documented to move thousands of kilometres, they are most frequently observed at a few predictable seasonal aggregation sites. The absence of sharks at the surface during visual surveys has led to the assumption that sharks disperse to places unknown during the long ‘off-seasons’ at most of these locations. Here we compare 2 years of R. typus visual sighting records from Mafia Island in Tanzania to concurrent acoustic telemetry of tagged individuals. Sightings revealed a clear seasonal pattern with a peak between October and February and no sharks observed at other times. By contrast, acoustic telemetry demonstrated year-round residency of R. typus. The sharks use a different habitat in the off-season, swimming deeper and further away from shore, presumably in response to prey distributions. This behavioural change reduces the sharks'' visibility, giving the false impression that they have left the area. We demonstrate, for the first time to our knowledge, year-round residency of unprovisioned, individual R. typus at an aggregation site, and highlight the importance of using multiple techniques to study the movement ecology of marine megafauna.     

Childhood Learning Disabilities and Atypical Dementia: A Retrospective Chart Review

Objective

To further our understanding of the association between self-reported childhood learning disabilities (LDs) and atypical dementia phenotypes (Atypical Dementia), including logopenic primary progressive aphasia (L-PPA), Posterior Cortical Atrophy (PCA), and Dysexecutive-type Alzheimer’s Disease (AD).

Methods

This retrospective case series analysis of 678 comprehensive neuropsychological assessments compared rates of self-reported LD between dementia patients diagnosed with Typical AD and those diagnosed with Atypical Dementia. 105 cases with neuroimaging or CSF data available and at least one neurology follow-up were identified as having been diagnosed by the neuropsychologist with any form of neurodegenerative dementia. These cases were subject to a consensus diagnostic process among three dementia experts using validated clinical criteria for AD and PPA. LD was considered Probable if two or more statements consistent with prior LD were documented within the Social & Developmental History of the initial neuropsychological evaluation.

Results

85 subjects (Typical AD n=68, Atypical AD n=17) were included in the final analysis. In logistic regression models adjusted for age, gender, handedness, education and symptom duration, patients with Probable LD, compared to patients without Probable LD, were significantly more likely to be diagnosed with Atypical Dementia vs. Typical AD (OR 13.1, 95% CI 1.3-128.4). All three of the L-PPA cases reporting a childhood LD endorsed childhood difficulty with language. By contrast, both PCA cases reporting Probable childhood LD endorsed difficulty with attention and/or math.

Conclusions

In people who develop dementia, childhood LD may predispose to atypical phenotypes. Future studies are required to confirm whether atypical neurodevelopment predisposes to regional-specific neuropathology in AD and other dementias.     

Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes

Here, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Förster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes. Furthermore, the data suggest that the V664E mutation causes a very small, but statistically significant change in dimer structure. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Labeling phospholipid membranes with lipid mimetic luminescent metal complexes

Lipid-mimetic metallosurfactant based luminophores are promising candidates for labeling phospholipid membranes without altering their biophysical characteristics. The metallosurfactants studied exhibit high structural and physicochemical similarity to phospholipid molecules, designed to incorporate into the membrane structure without the need for covalent attachment to a lipid molecule. In this work, two lipid-mimetic phosphorescent metal complexes are described: [Ru(bpy)₂(dn-bpy)]^2 + and [Ir(ppy)₂(dn-bpy)]⁺ where bpy is 2,2′-bipyridine, dn-bpy is 4,4′-dinonyl-2,2′-bipyridine and ppy is 2-phenylpyridine. Apart from being lipid-mimetic in size, shape and physical properties, both complexes exhibit intense photoluminescence and enhanced photostability compared with conventional organic fluorophores, allowing for prolonged observation. Moreover, the large Stokes shift and long luminescence lifetime associated with these complexes make them more suitable for spectroscopic studies. The complexes are easily incorporated into dimyristoil-phosphatidyl-choline (DMPC) liposomes by mixing in the organic solvent phase. DLS reveals the labeled membranes form liposomes of similar size to that of neat DMPC membrane. Synchrotron Small-Angle X-ray Scattering (SAXS) measurements confirmed that up to 5% of either complex could be incorporated into DMPC membranes without producing any structural changes in the membrane. Fluorescence microscopy reveals that 0.5% label content is sufficient for imaging. Atomic Force Microscopic imaging confirms that liposomes of the labeled bilayers on a mica surface can fuse into a flat lamellar membrane that is morphologically identical to neat lipid membranes. These results demonstrate the potential of such lipid-mimetic luminescent metal complexes as a new class of labels for imaging lipid membranes.     

Open and Closed Conformations of the Isolated Transmembrane Domain of Death Receptor 5 Support a New Model of Activation

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer—sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers—undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.Death receptor 5 (DR5) is a member of the tumor necrosis factor receptor (TNFR) superfamily that mediates apoptosis when bound by its cognate ligand, TNF-related apoptosis-inducing ligand (1). Upregulated in cancer cells, DR5 is among the most actively pursued anticancer targets (2). TNF-related apoptosis-inducing ligand binds to preassembled DR5 trimers at their extracellular domains, causing the formation of oligomeric ligand-receptor networks that are held together by receptor dimers (3). In the long-isoform of DR5, this dimer is crosslinked via ligand-induced disulfide bond formation between two transmembrane (TM) domain α-helices at Cys-209, and is further stabilized by a GxxxG motif one helix-turn downstream (3).Our recent study of the structurally homologous TNFR1 showed that receptor activation involves a conformational change that propagates from the extracellular domain to the cytosolic domain through a separation (or opening) of the TM domains of the dimer (4). We have therefore hypothesized that the activation of DR5, and indeed all structurally homologous TNF-receptors, involves a scissorslike opening of the TM domain dimer (Fig. 1).Open in a separate windowFigure 1Activation model of the DR5-L TM dimer. The sequence and positions of the disulfide bond and TOAC spin label (top), along with our previously published model (bottom, left) are shown. We propose an activation model (bottom, right) in which the transmembrane dimer pivots at its disulfide bond to reach an active open conformation.Using electron paramagnetic resonance (EPR) spectroscopy, a technique that has been used previously to study TM helix architecture and dynamics (5,6), and potential-of-mean force (PMF) calculations (7,8), this study addresses the question of whether the isolated disulfide-linked DR5-L TM domain dimer occupies distinct open and closed states (Fig. 1), and how its dynamic behavior contributes to the free-energy landscape of the opening transition of the full-length receptor.The DR5-L TM domain was synthesized with TOAC, an amino acid with a nitroxide spin label rigidly fixed to the α-carbon (9), incorporated at position 32 (Fig. 1), with some minor modification to facilitate EPR measurements. Previous work confirmed that this peptide forms disulfide-linked dimers (e.g., via comparison to 2-ME treated sample) and a negligible population of higher-order oligomers (further supported by model fitting of the EPR data below). For peptide work, residues were renumbered such that Thr-204 corresponds to Thr-1, and so on. The cytosolic Cys-29 (which we previously showed does not participate in a disulfide bond in cells) was replaced with serine to prevent the formation of antiparallel disulfide-linked dimers, and Trp-34 was replaced with tyrosine to prevent intrinsic fluorescence in fluorescence studies (not published). Continuous-wave (CW) dipolar EPR (sensitive only to spin-spin distances <25 Å) was used to measure TOAC-TOAC distances within the TM dimers and revealed an ordered Gaussian distribution centered at 16 Å (full width half-maximum (FWHM) = 4 Å), corresponding to a closed state (Fig. 2 A). Double electron-electron resonance (DEER) (sensitive to spin-spin distances from 15 to 60 Å) also detected a short distance consistent with the dipolar EPR data, along with a longer, disordered component (32.9 Å, FWHM = 28 Å) (Fig. 2 B). Together, these measurements indicate the presence of a compact, ordered closed state and a broader, disordered open state. EPR on oriented membranes also indicated two structural states. Global fitting revealed two populations of spin-label tilt angles (orientation of the nitroxide principal axis relative to the membrane normal): a narrow conformation (24°, FWHM = 20°), and a disordered conformation (50°, FWHM = 48°) (Fig. 2 C). This bimodal orientational distribution (Fig. 2 C) is remarkably consistent with the bimodal distance distribution (Fig. 2 B).Open in a separate windowFigure 2EPR spectra (left) of 32-TOAC-DR5 in lipid, and resulting structural distributions (right). (A) CW dipolar EPR spectra (left) of dimer (1 mM diamide) and monomer (1 mM 2-mercaptoethanol). Best-fit spin-spin distance distribution was a single Gaussian centered at 16 ± 2 Å (right). (B) The DEER waveform (left) of 32-TOAC-DR5 dimer was best fit (right) to a two-Gaussian distribution. The short distance was constrained to agree with the CW data, because DEER has poor sensitivity for distances <20 Å. The long-distance distribution is centered at 32.9 Å and is much broader. (C) CW EPR spectra (left) of 32-TOAC-DR5, with the membrane-normal oriented parallel (red) and perpendicular (blue) to the field. Simultaneous (global) fitting of these spectra reveals narrow and broad components (right). (In panels B and C, the overall distribution is plotted as black, while the closed and open components are plotted as green and magenta, respectively.)We subsequently conducted a PMF calculation (10) using the DR5-L TM dimer starting configuration developed by our group previously (3), embedded in a DMPC bilayer, with the Leu-32/Leu-32 C_α distance as the reaction coordinate. Three calculations were run from independent starting configurations, each using 50 windows spaced in 0.5° increments, and run for 20 ns at each window (totaling 3 μs). Each of the calculations yielded a similar result, and the averaged free energy curve (Fig. 3 A) agrees remarkably well with our EPR measurements: a narrow distribution at the closed conformation (∼16 Å, Fig. 3 B) separated by an ∼3 kcal/mol energy barrier from a broad distribution of accessible open conformations at ∼27 Å, (Fig. 3 C). Each of the three individual PMF plots can be found in Fig. S1 in the Supporting Material.Open in a separate windowFigure 3(A) PMF calculation of the DR5 TM domain dimer along the Leu-32/Leu-32 distance reaction coordinate. The PMF calculation reveals a narrow closed state and a broader open state separated by a free energy barrier. Representative snapshots of the (B) closed state and (C) open state.In the closed state, the helices are tightly packed at the GxxxG interfacial motif and all the way down the juxtaposed helix faces at residues Ala-18, Leu-22, Ala-25, and Val-26. The tight packing is aided by kinking and twisting of the two helices around their common axis, increasing the interacting surface area. In the open conformations, the Ala-18, Leu-22, Ala-25, and Val-26 pairs are dissociated and, interestingly, the GxxxG motif at Gly-10 and Gly-14 remains tightly packed. The open state energy well is only slightly less favorable than the closed state (by ∼2 kcal/mol), and its free energy profile is relatively broad and flat. The increased crossing angle in the open state is facilitated by straightening of the helix kink and is not accommodated by a change in bilayer thickness (see Fig. S3, A and B).The observed change in helix-helix distance (11 Å between the two minima in the PMF) is extremely close to that observed previously in live-cell FRET studies of a constitutively active form of TNFR1 (∼8 Å change between states using large fluorescence probes at the cytosolic domains) (4). The change observed in the EPR data (17 Å) may be an overestimate because the measurement is made between TOAC spin labels that likely protrude from the two helices, depending on rotational orientation. These results collectively show that activation of these receptors requires a small, but clearly significant conformational opening of the TM domains. One important note is that our EPR experiments recapitulate the equilibrium distribution of the two states despite there being no driving force to traverse the barrier between them (∼3 kcal/mol in the closed-to-open transition and ∼1 kcal/mol in the open-to-closed transition, Fig. 3). We do not interpret the results to mean that the dimer necessarily traverses these barriers at 4°C. Rather, there likely exist multiple reaction paths for dimerization of the abstracted TM domains. Finally, in the context of the full-length receptor, how the ligand induces a conformational change capable of overcoming the closed-to-open barrier remains an important question.Whether the observed structural transition in the TM domain dimer of the long-isoform of DR5 is a ubiquitous conformational switch that acts over the entire TNFR superfamily remains unknown. Vilar et al. (11) first proposed a similar scissors-model for activation of p75 neurotrophin receptor, which has a cysteine at the center of its TM helix. The short isoform of DR5 lacks a TM domain cysteine, but does form noncovalent dimers in cells, with likely TM domain dimer contacts (3). Among the other closely related and structurally homologous members of the TNFR superfamily, TNFR1 contains a cysteine at the center of the TM domain, but lacks any discernible small residue motifs (e.g., GxxxG). TNFR2 lacks a TM cysteine on the extracellular side, but does have a GxxxG motif positioned similarly to that of DR5. On the other hand, Death Receptor 4, whose functional distinction from DR5 has remained somewhat elusive, lacks both a cysteine and any recognizable small-residue hydrophobic motif.In summary, we have extended recent findings that point to the TM domain of DR5 as an essential structural component in the conformational change associated with activation. Our findings that the DR5-L TM domain occupies distinct open and closed states, separated by a substantial energy barrier, points the way to further studies across the TNF-receptor superfamily.