ResearchPad - ion-channels https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Impact of intracellular hemin on N-type inactivation of voltage-gated K<sup>+</sup> channels]]> https://www.researchpad.co/article/elastic_article_9972 N-type inactivation of voltage-gated K+ channels is conferred by the N-terminal “ball” domains of select pore-forming α subunits or of auxiliary β subunits, and influences electrical cellular excitability. Here, we show that hemin impairs inactivation of K+ channels formed by Kv3.4 α subunits as well as that induced by the subunits Kvβ1.1, Kvβ1.2, and Kvβ3.1 when coexpressed with α subunits of the Kv1 subfamily. In Kvβ1.1, hemin interacts with cysteine and histidine residues in the N terminus (C7 and H10) with high affinity (EC50 100 nM). Similarly, rapid inactivation of Kv4.2 channels induced by the dipeptidyl peptidase-like protein DPP6a is also sensitive to hemin, and the DPP6a mutation C13S eliminates this dependence. The results suggest a common mechanism for a dynamic regulation of Kv channel inactivation by heme/hemin in N-terminal ball domains of Kv α and auxiliary β subunits. Free intracellular heme therefore has the potential to regulate cellular excitability via modulation of Kv channel inactivation.

Electronic supplementary materialThe online version of this article (10.1007/s00424-020-02386-1) contains supplementary material, which is available to authorized users. ]]>
<![CDATA[Model based estimation of QT intervals in non-invasive fetal ECG signals]]> https://www.researchpad.co/article/elastic_article_7659 The end timing of T waves in fetal electrocardiogram (fECG) is important for the evaluation of ST and QT intervals which are vital markers to assess cardiac repolarization patterns. Monitoring malignant fetal arrhythmias in utero is fundamental to care in congenital heart anomalies preventing perinatal death. Currently, reliable detection of end of T waves is possible only by using fetal scalp ECG (fsECG) and fetal magnetocardiography (fMCG). fMCG is expensive and less accessible and fsECG is an invasive technique available only during intrapartum period. Another safer and affordable alternative is the non-invasive fECG (nfECG) which can provide similar assessment provided by fsECG and fMECG but with less accuracy (not beat by beat). Detection of T waves using nfECG is challenging because of their low amplitudes and high noise. In this study, a novel model-based method that estimates the end of T waves in nfECG signals is proposed. The repolarization phase has been modeled as the discharging phase of a capacitor. To test the model, fECG signals were collected from 58 pregnant women (age: (34 ± 6) years old) bearing normal and abnormal fetuses with gestational age (GA) 20-41 weeks. QT and QTc intervals have been calculated to test the level of agreement between the model-based and reference values (fsECG and Doppler Ultrasound (DUS) signals) in normal subjects. The results of the test showed high agreement between model-based and reference values (difference < 5%), which implies that the proposed model could be an alternative method to detect the end of T waves in nfECG signals.

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<![CDATA[Influence of the tubular network on the characteristics of calcium transients in cardiac myocytes]]> https://www.researchpad.co/article/N7f446290-780e-4486-a1de-95187c6060a1

Transverse and axial tubules (TATS) are an essential ingredient of the excitation-contraction machinery that allow the effective coupling of L-type Calcium Channels (LCC) and ryanodine receptors (RyR2). They form a regular network in ventricular cells, while their presence in atrial myocytes is variable regionally and among animal species We have studied the effect of variations in the TAT network using a bidomain computational model of an atrial myocyte with variable density of tubules. At each z-line the t-tubule length is obtained from an exponential distribution, with a given mean penetration length. This gives rise to a distribution of t-tubules in the cell that is characterized by the fractional area (F.A.) occupied by the t-tubules. To obtain consistent results, we average over different realizations of the same mean penetration length. To this, in some simulations we add the effect of a network of axial tubules. Then we study global properties of calcium signaling, as well as regional heterogeneities and local properties of sparks and RyR2 openings. In agreement with recent experiments in detubulated ventricular and atrial cells, we find that detubulation reduces the calcium transient and synchronization in release. However, it does not affect sarcoplasmic reticulum (SR) load, so the decrease in SR calcium release is due to regional differences in Ca2+ release, that is restricted to the cell periphery in detubulated cells. Despite the decrease in release, the release gain is larger in detubulated cells, due to recruitment of orphaned RyR2s, i.e, those that are not confronting a cluster of LCCs. This probably provides a safeguard mechanism, allowing physiological values to be maintained upon small changes in the t-tubule density. Finally, we do not find any relevant change in spark properties between tubulated and detubulated cells, suggesting that the differences found in experiments could be due to differential properties of the RyR2s in the membrane and in the t-tubules, not incorporated in the present model. This work will help understand the effect of detubulation, that has been shown to occur in disease conditions such as heart failure (HF) in ventricular cells, or atrial fibrillation (AF) in atrial cells.

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<![CDATA[Taking a close look at a large-pore channel]]> https://www.researchpad.co/article/Ne1f3b2bd-883f-41cc-b649-888e9e881f84

The structure of pannexin 1, a channel protein with a large pore, has been determined for the first time.

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<![CDATA[β-blockers after acute myocardial infarction in patients with chronic obstructive pulmonary disease: A nationwide population-based observational study]]> https://www.researchpad.co/article/5c8823d2d5eed0c4846390b1

Background

Patients with chronic obstructive pulmonary disease (COPD) less often receive β-blockers after acute myocardial infarction (AMI). This may influence their outcomes after AMI. This study evaluated the efficacy of β-blockers after AMI in patients with COPD, compared with non-dihydropyridine calcium channel blockers (NDCCBs) and absence of these two kinds of treatment.

Methods and results

We conducted a nationwide population-based cohort study using data retrieved from Taiwan National Health Insurance Research Database. We collected 28,097 patients with COPD who were hospitalized for AMI between January 2004 and December 2013. After hospital discharge, 24,056 patients returned to outpatient clinics within 14 days (the exposure window). Those who received both β-blockers and NDCCBs (n = 302) were excluded, leaving 23,754 patients for analysis. Patients were classified into the β-blocker group (n = 10,638, 44.8%), the NDCCB group, (n = 1,747, 7.4%) and the control group (n = 11,369, 47.9%) based on their outpatient prescription within the exposure window. The β-blockers group of patients had lower overall mortality risks (adjusted hazard ratio [95% confidence interval]: 0.91 [0.83–0.99] versus the NDCCB group; 0.88 [0.84–0.93] versus the control group), but the risk of major adverse cardiac events within 1 year was not statistically different. β-blockers decreased risks of re-hospitalization for COPD and other respiratory diseases by 12–32%.

Conclusions

The use of β-blockers after AMI was associated with a reduced mortality risk in patients with COPD. β-blockers did not increase the risk of COPD exacerbations.

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<![CDATA[Trpm4 ion channels in pre-Bötzinger complex interneurons are essential for breathing motor pattern but not rhythm]]> https://www.researchpad.co/article/5c784fa8d5eed0c484007252

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca2+)-activated nonselective cationic current (ICAN) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing ICAN is predicted to slow down or stop breathing; its contributions to motor pattern would be reflected in the magnitude of movements (output). We tested the role(s) of ICAN using reverse genetic techniques to diminish its putative ion channels Trpm4 or Trpc3 in preBötC neurons in vivo. Adult mice transduced with Trpm4-targeted short hairpin RNA (shRNA) progressively decreased the tidal volume of breaths yet surprisingly increased breathing frequency, often followed by gasping and fatal respiratory failure. Mice transduced with Trpc3-targeted shRNA survived with no changes in breathing. Patch-clamp and field recordings from the preBötC in mouse slices also showed an increase in the frequency and a decrease in the magnitude of preBötC neural bursts in the presence of Trpm4 antagonist 9-phenanthrol, whereas the Trpc3 antagonist pyrazole-3 (pyr-3) showed inconsistent effects on magnitude and no effect on frequency. These data suggest that Trpm4 mediates ICAN, whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated ICAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator.

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<![CDATA[Using computational models to predict in vivo synaptic inputs to interneuron specific 3 (IS3) cells of CA1 hippocampus that also allow their recruitment during rhythmic states]]> https://www.researchpad.co/article/5c3e50b5d5eed0c484d84993

Brain coding strategies are enabled by the balance of synaptic inputs that individual neurons receive as determined by the networks in which they reside. Inhibitory cell types contribute to brain function in distinct ways but recording from specific, inhibitory cell types during behaviour to determine their contributions is highly challenging. In particular, the in vivo activities of vasoactive intestinal peptide-expressing interneuron specific 3 (IS3) cells in the hippocampus that only target other inhibitory cells are unknown at present. We perform a massive, computational exploration of possible synaptic inputs to IS3 cells using multi-compartment models and optimized synaptic parameters. We find that asynchronous, in vivo-like states that are sensitive to additional theta-timed inputs (8 Hz) exist when excitatory and inhibitory synaptic conductances are approximately equally balanced and with low numbers of activated synapses receiving correlated inputs. Specifically, under these balanced conditions, the input resistance is larger with higher mean spike firing rates relative to other activated synaptic conditions investigated. Incoming theta-timed inputs result in strongly increased spectral power relative to baseline. Thus, using a generally applicable computational approach we predict the existence and features of background, balanced states in hippocampal circuits.

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<![CDATA[Mechanistic insight into spontaneous transition from cellular alternans to arrhythmia—A simulation study]]> https://www.researchpad.co/article/5c0ae43ed5eed0c484589394

Cardiac electrical alternans (CEA), manifested as T-wave alternans in ECG, is a clinical biomarker for predicting cardiac arrhythmias and sudden death. However, the mechanism underlying the spontaneous transition from CEA to arrhythmias remains incompletely elucidated. In this study, multiscale rabbit ventricular models were used to study the transition and a potential role of INa in perpetuating such a transition. It was shown CEA evolved into either concordant or discordant action potential (AP) conduction alternans in a homogeneous one-dimensional tissue model, depending on tissue AP duration and conduction velocity (CV) restitution properties. Discordant alternans was able to cause conduction failure in the model, which was promoted by impaired sodium channel with either a reduced or increased channel current. In a two-dimensional homogeneous tissue model, a combined effect of rate- and curvature-dependent CV broke-up alternating wavefronts at localised points, facilitating a spontaneous transition from CEA to re-entry. Tissue inhomogeneity or anisotropy further promoted break-up of re-entry, leading to multiple wavelets. Similar observations have also been seen in human atrial cellular and tissue models. In conclusion, our results identify a mechanism by which CEA spontaneously evolves into re-entry without a requirement for premature ventricular complexes or pre-existing tissue heterogeneities, and demonstrated the important pro-arrhythmic role of impaired sodium channel activity. These findings are model-independent and have potential human relevance.

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<![CDATA[Compartment models for the electrical stimulation of retinal bipolar cells]]> https://www.researchpad.co/article/5c215132d5eed0c4843f91fe

Bipolar cells of the retina are among the smallest neurons of the nervous system. For this reason, compared to other neurons, their delay in signaling is minimal. Additionally, the small bipolar cell surface combined with the low membrane conductance causes very little attenuation in the signal from synaptic input to the terminal. The existence of spiking bipolar cells was proven over the last two decades, but until now no complete model including all important ion channel types was published. The present study amends this and analyzes the impact of the number of model compartments on simulation accuracy. Characteristic features like membrane voltages and spike generation were tested and compared for one-, two-, four- and 117-compartment models of a macaque bipolar cell. Although results were independent of the compartment number for low membrane conductances (passive membranes), nonlinear regimes such as spiking required at least a separate axon compartment. At least a four compartment model containing the functionally different segments dendrite, soma, axon and terminal was needed for understanding signaling in spiking bipolar cells. Whereas for intracellular current application models with small numbers of compartments showed quantitatively correct results in many cases, the cell response to extracellular stimulation is sensitive to spatial variation of the electric field and accurate modeling therefore demands for a large number of short compartments even for passive membranes.

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<![CDATA[Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes]]> https://www.researchpad.co/article/5c1c0acbd5eed0c484426add

Calcium (Ca2+) is an important ion in physiology and is found both outside and inside cells. The intracellular concentration of Ca2+ is tightly regulated as it is an intracellular signal molecule and can affect a variety of cellular processes. In immune cells Ca2+ has been shown to regulate e.g. gene transcription, cytokine secretion, proliferation and migration. Ca2+ can enter the cytoplasm either from intracellular stores or from outside the cells when Ca2+ permeable ion channels in the plasma membrane open. The Ca2+ release-activated (CRAC) channel is the most prominent Ca2+ ion channel in the plasma membrane. It is formed by ORAI1-3 and the channel is opened by the endoplasmic reticulum Ca2+ sensor proteins stromal interaction molecules (STIM) 1 and 2. Another group of Ca2+ channels in the plasma membrane are the voltage-gated Ca2+ (CaV) channels. We examined if a change in immunological tolerance is accompanied by altered ORAI, STIM and CaV gene expression in peripheral blood mononuclear cells (PBMCs) in pregnant women and in type 1 diabetic individuals. Our results show that in pregnancy and type 1 diabetes ORAI1-3 are up-regulated whereas STIM1 and 2 are down-regulated in pregnancy but only STIM2 in type 1 diabetes. Expression of L-, P/Q-, R- and T-type voltage-gated Ca2+ channels was detected in the PBMCs where the CaV2.3 gene was up-regulated in pregnancy and type 1 diabetes whereas the CaV 2.1 and CaV3.2 genes were up-regulated only in pregnancy and the CaV1.3 gene in type 1 diabetes. The results are consistent with that expression of ORAI, STIM and CaV genes correlate with a shift in immunological status of the individual in health, as during pregnancy, and in the autoimmune disease type 1 diabetes. Whether the changes are in general protective or in type 1 diabetes include some pathogenic components remains to be clarified.

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<![CDATA[Prediction of perturbed proton transfer networks]]> https://www.researchpad.co/article/5c1ab879d5eed0c4840282dc

The transfer of protons through proton translocating channels is a complex process, for which direct samplings of different protonation states and side chain conformations in a transition network calculation provide an efficient, bias-free description. In principle, a new transition network calculation is required for every unsampled change in the system of interest, e.g. an unsampled protonation state change, which is associated with significant computational costs. Transition networks void of or including an unsampled change are termed unperturbed or perturbed, respectively. Here, we present a prediction method, which is based on an extensive coarse-graining of the underlying transition networks to speed up the calculations. It uses the minimum spanning tree and a corresponding sensitivity analysis of an unperturbed transition network as initial guess and refinement parameter for the determination of an unknown, perturbed transition network. Thereby, the minimum spanning tree defines a sub-network connecting all nodes without cycles and minimal edge weight sum, while the sensitivity analysis analyzes the stability of the minimum spanning tree towards individual edge weight reductions. Using the prediction method, we are able to reduce the calculation costs in a model system by up to 80%, while important network properties are maintained in most predictions.

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<![CDATA[Binding of the general anesthetic sevoflurane to ion channels]]> https://www.researchpad.co/article/5c059df1d5eed0c4849c981e

The direct-site hypothesis assumes general anesthetics bind ion channels to impact protein equilibrium and function, inducing anesthesia. Despite advancements in the field, a first principle all-atom demonstration of this structure-function premise is still missing. We focus on the clinically used sevoflurane interaction to anesthetic-sensitive Kv1.2 mammalian channel to resolve if sevoflurane binds protein’s well-characterized open and closed structures in a conformation-dependent manner to shift channel equilibrium. We employ an innovative approach relying on extensive docking calculations and free-energy perturbation of all potential binding sites revealed by the latter, and find sevoflurane binds open and closed structures at multiple sites under complex saturation and concentration effects. Results point to a non-trivial interplay of site and conformation-dependent modes of action involving distinct binding sites that increase channel open-probability at diluted ligand concentrations. Given the challenge in exploring more complex processes potentially impacting channel-anesthetic interaction, the result is revealing as it demonstrates the process of multiple anesthetic binding events alone may account for open-probability shifts recorded in measurements.

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<![CDATA[A sensory-motor neuron type mediates proprioceptive coordination of steering in C. elegans via two TRPC channels]]> https://www.researchpad.co/article/5b28b93c463d7e146ff345d4

Animal locomotion is mediated by a sensory system referred to as proprioception. Defects in the proprioceptive coordination of locomotion result in uncontrolled and inefficient movements. However, the molecular mechanisms underlying proprioception are not fully understood. Here, we identify two transient receptor potential cation (TRPC) channels, trp-1 and trp-2, as necessary and sufficient for proprioceptive responses in C. elegans head steering locomotion. Both channels are expressed in the SMDD neurons, which are required and sufficient for head bending, and mediate coordinated head steering by sensing mechanical stretches due to the contraction of head muscle and orchestrating dorsal head muscle contractions. Moreover, the SMDD neurons play dual roles to sense muscle stretch as well as to control muscle contractions. These results demonstrate that distinct locomotion patterns require dynamic and homeostatic modulation of feedback signals between neurons and muscles.

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<![CDATA[Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells]]> https://www.researchpad.co/article/5989da16ab0ee8fa60b7b622

The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.

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<![CDATA[Correlation of Apical Fluid-Regulating Channel Proteins with Lung Function in Human COPD Lungs]]> https://www.researchpad.co/article/5989da9bab0ee8fa60ba3dc9

Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

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<![CDATA[The Role of Glutamate Release on Voltage-Dependent Anion Channels (VDAC)-Mediated Apoptosis in an Eleven Vessel Occlusion Model in Rats]]> https://www.researchpad.co/article/5989d9d5ab0ee8fa60b65905

Voltage-dependent anion channel (VDAC) is the main protein in mitochondria-mediated apoptosis, and the modulation of VDAC may be induced by the excessive release of extracellular glutamate. This study examined the role of glutamate release on VDAC-mediated apoptosis in an eleven vessel occlusion model in rats. Male Sprague-Dawley rats (250–350 g) were used for the 11 vessel occlusion ischemic model, which were induced for a 10-min transient occlusion. During the ischemic and initial reperfusion episode, the real-time monitoring of the extracellular glutamate concentration was measured using an amperometric microdialysis biosensor and the cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry. To confirm neuronal apoptosis, the brains were removed 72 h after ischemia to detect the neuron-specific nuclear protein and pro-apoptotic proteins (cleaved caspase-3, VDAC, p53 and BAX). The changes in the mitochondrial morphology were measured by atomic force microscopy. A decrease in the % of CBF was observed, and an increase in glutamate release was detected after the onset of ischemia, which continued to increase during the ischemic period. A significantly higher level of glutamate release was observed in the ischemia group. The increased glutamate levels in the ischemia group resulted in the activation of VDAC and pro-apoptotic proteins in the hippocampus with morphological alterations to the mitochondria. This study suggests that an increase in glutamate release promotes VDAC-mediated apoptosis in an 11 vessel occlusion ischemic model.

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<![CDATA[RNA-Seq Transcriptome Analysis of Direction-Selective T4/T5 Neurons in Drosophila]]> https://www.researchpad.co/article/5989dabaab0ee8fa60bae4d9

Neuronal computation underlying detection of visual motion has been studied for more than a half-century. In Drosophila, direction-selective T4/T5 neurons show supralinear signal amplification in response to stimuli moving in their preferred direction, in agreement with the prediction made by the Hassenstein-Reichardt detector. Nevertheless, the molecular mechanism explaining how the Hassenstein-Reichardt model is implemented in T4/T5 cells has not been identified yet. In the present study, we utilized cell type-specific transcriptome profiling with RNA-seq to obtain a complete gene expression profile of T4/T5 neurons. We analyzed the expression of genes that affect neuronal computational properties and can underlie the molecular implementation of the core features of the Hassenstein-Reichardt model to the dendrites of T4/T5 neurons. Furthermore, we used the acquired RNA-seq data to examine the neurotransmitter system used by T4/T5 neurons. Surprisingly, we observed co-expression of the cholinergic markers and the vesicular GABA transporter in T4/T5 neurons. We verified the previously undetected expression of vesicular GABA transporter in T4/T5 cells using VGAT-LexA knock-in line. The provided gene expression dataset can serve as a useful source for studying the properties of direction-selective T4/T5 neurons on the molecular level.

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<![CDATA[The Lethal Toxin from Australian Funnel-Web Spiders Is Encoded by an Intronless Gene]]> https://www.researchpad.co/article/5989daf2ab0ee8fa60bc1bb1

Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome.

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<![CDATA[Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues]]> https://www.researchpad.co/article/5989da0cab0ee8fa60b781b4

Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies.

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<![CDATA[Pharmacological Activation/Inhibition of the Cannabinoid System Affects Alcohol Withdrawal-Induced Neuronal Hypersensitivity to Excitotoxic Insults]]> https://www.researchpad.co/article/5989da38ab0ee8fa60b86fd4

Cessation of chronic ethanol consumption can increase the sensitivity of the brain to excitotoxic damages. Cannabinoids have been proposed as neuroprotectants in different models of neuronal injury, but their effect have never been investigated in a context of excitotoxicity after alcohol cessation. Here we examined the effects of the pharmacological activation/inhibition of the endocannabinoid system in an in vitro model of chronic ethanol exposure and withdrawal followed by an excitotoxic challenge. Ethanol withdrawal increased N-methyl-D-aspartate (NMDA)-evoked neuronal death, probably by altering the ratio between GluN2A and GluN2B NMDA receptor subunits. The stimulation of the endocannabinoid system with the cannabinoid agonist HU-210 decreased NMDA-induced neuronal death exclusively in ethanol-withdrawn neurons. This neuroprotection could be explained by a decrease in NMDA-stimulated calcium influx after the administration of HU-210, found exclusively in ethanol-withdrawn neurons. By contrast, the inhibition of the cannabinoid system with the CB1 receptor antagonist rimonabant (SR141716) during ethanol withdrawal increased death of ethanol-withdrawn neurons without any modification of NMDA-stimulated calcium influx. Moreover, chronic administration of rimonabant increased NMDA-stimulated toxicity not only in withdrawn neurons, but also in control neurons. In summary, we show for the first time that the stimulation of the endocannabinoid system is protective against the hyperexcitability developed during alcohol withdrawal. By contrast, the blockade of the endocannabinoid system is highly counterproductive during alcohol withdrawal.

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