Open Channel Block of HERG K ϩ Channels by Vesnarinone

ABSTRACT Vesnarinone, a cardiotonic agent, blocks I Kr and, unlike other I Kr blockers, produces a frequency-dependent prolongation of action potential duration (APD). To elucidate the mechanisms, we studied the effects of vesnarinone on HERG, the cloned human I Kr channel, heterologously expressed in Xenopus laevis oocytes. Vesnarinone caused a concentration-dependent inhibition of HERG currents with an IC 50 value of 17.7 Ϯ 2.5 M at 0 mV (n ϭ 6). When HERG was coexpressed with the ␤-subunit MiRP1, a similar potency for block was measured (IC 50 : 15.0 Ϯ 3.0 M at 0 mV, n ϭ 5). Tonic block of the HERG channel current was minimal (Ͻ5% at 30 M, n ϭ 5). The rate of onset of block and the steady-state value for block of current were not significantly different for test potentials ranging from Ϫ40 to ϩ40 mV [time constant () ϭ 372 Ϯ 76 ms at ϩ40 mV, n ϭ 4]. Recovery from block at Ϫ60, Ϫ90, and Ϫ120 mV was not significantly different ( ϭ 8.5 Ϯ 1.5 s at Ϫ90 mV, n ϭ 4). Vesnarinone produced similar effects on inactivation-removed mutant (G628C/S631C) HERG channels. The IC 50 value was 10.7 Ϯ 3.7 M at 0 mV (n ϭ 5), and the onset and recovery from block of current findings were similar to those of wild-type HERG. Amino acids important for the binding of vesnarinone were identified using alanine-scanning mutagenesis of residues believed to line the inner cavity of the HERG channel. Six important residues were identified, including G648, F656, and V659 located in the S6 domain and T623, S624, and V625 located at the base of the pore helix. These residues are similar but not identical to those determined previously for MK-499, an antiarrhythmic drug. In conclusion, vesnarinone preferentially blocks open HERG channels, with little effect on channels in the rested or inactivated state. These actions may contribute to the favorable frequency-dependent prolongation in APD

A Novel Mechanism for Calmodulin Dependent Inactivation of Transient Receptor Potential Vanilloid 6

The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the Transient Receptor Potential (TRP) superfamily of ion channels and both play an important role in overall Cahomeostasis. The functioning of the channels centres on a tightly controlled Ca-dependent feedback mechanism where the direct binding of the universal Ca-binding protein calmodulin (CaM) to the channel's C-terminal tail is required for channel inactivation. We have investigated this interaction at the atomic level and propose that under basal cellular [CaCaM is constitutively bound to the channel's C-tail via CaM C-lobe only contacts. When cytosolic [Ca] increases charging the apo CaM N-lobe with Ca, the CaM:TRPV6 complex rearranges and the TRPV6 C-tail further engages the CaM N-lobe via a crucial interaction involving L707. In a cellular context, mutation of L707 significantly increased the rate of channel inactivation. Finally, we present a model for TRPV6 CaM-dependent inactivation, which involves a novel so-called "two-tail" mechanism whereby CaM bridges between two TRPV6 monomers resulting in closure of the channel pore

New Pyrimido-Indole Compound CD-160130 Preferentially Inhibits the K V

K(V)11.1 (hERG1) channels are often overexpressed in human cancers. In leukemias, K(V)11.1 regulates pro-survival signals that promote resistance to chemotherapy, raising the possibility that inhibitors of K(V)11.1 could be therapeutically beneficial. However, because of the role of K(V)11.1 in cardiac repolarization, blocking these channels may cause cardiac arrhythmias. We show that CD-160130, a novel pyrimido-indole compound, blocks K(V)11.1 channels with a higher efficacy for the K(V)11.1 isoform B, in which the IC50 (1.8 mu M) was approximately 10-fold lower than observed in K(V)11.1 isoform A. At this concentration, CD-160130 also had minor effects on K(ir)2.1, K-V 1.3, K(v)1.5, and K(Ca)3.1. In vitro, CD-160130 induced leukemia cell apoptosis, and could overcome bone marrow mesenchymal stromal cell (MSC)-induced chemoresistance. This effect was caused by interference with the survival signaling pathways triggered by MSCs. In vivo, CD-160130 produced an antileukemic activity, stronger than that caused by cytarabine. Consistent with its atypical target specificity, CD-160130 did not bind to the main binding site of the arrhythmogenic K(V)11.1 blockers (the Phe656 pore residue). Importantly, in guinea pigs CD-160130 produced neither alteration of the cardiac action potential shape in dissociated cardiomyocytes nor any lengthening of the QT interval in vivo. Moreover, CD-160130 had no myelotoxicity on human bone marrow-derived cells. Therefore, CD-160130 is a promising first-in-class compound to attempt oncologic therapy without cardiotoxicity, based on targeting K(V)11.1. Because leukemia and cardiac cells tend to express different ratios of the A and B K(V)11.1 isoforms, the pharmacological properties of CD-160130 may depend, at least in part, on isoform specificity

Population Structure and Phylogeography in Nassau Grouper (Epinephelus striatus), a Mass-Aggregating Marine Fish

To address patterns of genetic connectivity in a mass-aggregating marine fish, we analyzed genetic variation in mitochondrial DNA (mtDNA), microsatellites, and single nucleotide polymorphisms (SNPs) for Nassau grouper (Epinephelus striatus). We expected Nassau grouper to exhibit genetic differentiation among its subpopulations due to its reproductive behavior and retentive oceanographic conditions experienced across the Caribbean basin. All samples were genotyped for two mitochondrial markers and 9 microsatellite loci, and a subset of samples were genotyped for 4,234 SNPs. We found evidence of genetic differentiation in a Caribbean-wide study of this mass-aggregating marine fish using mtDNA (FST = 0.206, p<0.001), microsatellites (FST = 0.002, p = 0.004) and SNPs (FST = 0.002, p = 0.014), and identified three potential barriers to larval dispersal. Genetically isolated regions identified in our work mirror those seen for other invertebrate and fish species in the Caribbean basin. Oceanographic regimes in the Caribbean may largely explain patterns of genetic differentiation among Nassau grouper subpopulations. Regional patterns observed warrant standardization of fisheries management and conservation initiatives among countries within genetically isolated regions

Testing a global standard for quantifying species recovery and assessing conservation impact.

Recognizing the imperative to evaluate species recovery and conservation impact, in 2012 the International Union for Conservation of Nature (IUCN) called for development of a "Green List of Species" (now the IUCN Green Status of Species). A draft Green Status framework for assessing species' progress toward recovery, published in 2018, proposed 2 separate but interlinked components: a standardized method (i.e., measurement against benchmarks of species' viability, functionality, and preimpact distribution) to determine current species recovery status (herein species recovery score) and application of that method to estimate past and potential future impacts of conservation based on 4 metrics (conservation legacy, conservation dependence, conservation gain, and recovery potential). We tested the framework with 181 species representing diverse taxa, life histories, biomes, and IUCN Red List categories (extinction risk). Based on the observed distribution of species' recovery scores, we propose the following species recovery categories: fully recovered, slightly depleted, moderately depleted, largely depleted, critically depleted, extinct in the wild, and indeterminate. Fifty-nine percent of tested species were considered largely or critically depleted. Although there was a negative relationship between extinction risk and species recovery score, variation was considerable. Some species in lower risk categories were assessed as farther from recovery than those at higher risk. This emphasizes that species recovery is conceptually different from extinction risk and reinforces the utility of the IUCN Green Status of Species to more fully understand species conservation status. Although extinction risk did not predict conservation legacy, conservation dependence, or conservation gain, it was positively correlated with recovery potential. Only 1.7% of tested species were categorized as zero across all 4 of these conservation impact metrics, indicating that conservation has, or will, play a role in improving or maintaining species status for the vast majority of these species. Based on our results, we devised an updated assessment framework that introduces the option of using a dynamic baseline to assess future impacts of conservation over the short term to avoid misleading results which were generated in a small number of cases, and redefines short term as 10 years to better align with conservation planning. These changes are reflected in the IUCN Green Status of Species Standard

An investigation of the mechanisms of cellular transformation by hERG potassium channels

Human ether-à-go-go-related gene 1 (hERG1) potassium channels are expressed in a variety of tumour cells and expression of hERG1 K+ channels in normal cells can induce a transformed phenotype. The transformative potential of hERG1 appears to be extracellular matrix-dependent. hERG1-expressing NIH-3T3 cells maintained a normal cell morphology when plated on collagen-1 and cell migration speeds were not different to those measured for empty vector-transfected NIH-3T3 cells (NIH-VC). However, hERG1-expressing NIH-3T3 cells displayed a transformed morphology and enhanced cell migration speeds when plated on laminin-1 or fibronectin, and this was associated with a reduction in vinculin protein cell content and cytoskeletal rearrangements. I have provided evidence to indicate that the ion flux through the hERG1 pore and its cell-surface localization is important for its oncogenic potential. Unlike for wild-type hERG1, stable expression of a non-conducting G628S hERG1, or a trafficking-deficient A561V hERG1 mutant did not induce a transformed phenotype in NIH-3T3 cells. Pentamidine, a compound which inhibits hERG1 trafficking to the cell-surface, inhibited fibronectin-dependent migration of wild-type hERG1-expressing cells. Although dofetilide, which blocks the ion conductance of hERG1, did not alter the transformative effect of wild-type hERG1 expression in cell grown on fibronectin, chronic application of this hERG1 inhibitor at a therapeutically-relevant concentration (100 nM) did cause a near-complete reversion of hERG1-expressing cells to a normal cell phenotype within 14 days. NIH-3T3 cells transiently transfected with a plasmid encoding both hERG1 and hERG1b exhibited increases in cell proliferation relative to cells expressing either isoform alone, suggesting a potential role for the hERG1b isoform in regulating hERG1 pro-oncogenic effects. In summary, the transforming potential of hERG1 expression appears to be dependent on hERG1 trafficking to the cell-surface and its ion channel functionality. Chronic administration of hERG1-blockers may be able to impair oncogenic progression in hERG1-expressing tumours.EThOS - Electronic Theses Online ServiceScholarhip - Mansoura UniversityEgyptGBUnited Kingdo

Memory-focused cognitive therapy for cocaine use disorder:Rationale, design and protocol for an external pilot randomised controlled trial

Introduction: Cocaine use disorder (CUD) is a debilitating condition characterised by maladaptive cocaine-related memories and impaired cognitive and behavioural control. There are no evidence-supported pharmacotherapies and only weakly effective psychological interventions specific for CUD. Our novel Memory-focused Cognitive Therapy (MFCT) aims to modify cocaine-related memories to reduce craving and drug use. Methods: This is a single-centre (outpatient), 15-week, two-arm, pilot randomised controlled trial (RCT) to address feasibility, safety, quality and preliminary efficacy. Thirty participants (adults ≥18 years; current CUD) will receive ongoing standard care (treatment-as-usual [TAU]) during the study and will be randomised (1:1) to a control or intervention group. The control group will receive 3 × 90min CUD cognitive case conceptualisation assessments and 2 × 30min cocaine-related cue-induction procedures (in vivo presentation of images and objects). Experimental group participants will receive 3 × 90min CUD cognitive case conceptualisation assessments; 2 × 30min cue-induction procedures; and individual MFCT (5 × 120min; daily for 1 week; with 3 relapse prevention follow-ups over 3-months). All study participants will complete research follow-ups at 1-week, 1-month and 3-months. The experimental and control groups will be compared on the mean score on the frequency version of the Craving Experience Questionnaire at 1-month (primary outcome measure). Secondary outcomes include: percentage of days abstinent and longest period of continuous abstinence from cocaine (past 28-days at 1-month follow-up); urine drug screen and CUD diagnosis (DSM-5). Conclusions: We will conduct a full external pilot RCT of a novel, MFCT for CUD. The findings will inform the case, and necessary modifications, for a substantive study