Channel Open Time Research Articles

A Feature Selection-Incorporated Simulation Study to Reveal the Effect of Calcium Ions on Cardiac Repolarization Alternans during Myocardial Ischemia

(1) Background: The main factors and their interrelationships contributing to cardiac repolarization alternans (CRA) remain unclear. This study aimed to elucidate the calcium (Ca2+)-related mechanisms underlying myocardial ischemia (MI)-induced CRA. (2) Materials and Methods: CRA was induced using S1 stimuli for pacing in an in silico ventricular model with MI. The standard deviations of nine Ca2+-related subcellular parameters among heartbeats from 100 respective nodes with and without alternans were chosen as features, including the maximum systole and end-diastole and corresponding differences in the Ca2+ concentration in the intracellular region([Ca2+]i) and junctional sarcoplasmic reticulum ([Ca2+]jsr), as well as the maximum opening of the L-type Ca2+ current (ICaL) voltage-dependent activation gate (d-gate), maximum closing of the inactivation gate (ff-gate), and the gated channel opening time (GCOT). Feature selection was applied to determine the importance of these features. (3) Results: The major parameters affecting CRA were the differences in [Ca2+]i at end-diastole, followed by the extent of d-gate activation and GCOT among beats. (4) Conclusions: MI-induced CRA is primarily characterized by functional changes in Ca2+ re-uptake, leading to alternans of [Ca2+]i and subsequent alternans of ICaL-dependent properties. The combination of computational simulation and machine learning shows promise in researching the underlying mechanisms of cardiac electrophysiology.

Salts of short-chain fatty acids increase the activity of the large conductance, Ca<sup>2+</sup>-activated K<sup>+</sup> channels and reduce calcium oscillations in rat GH3 cells

The short-chain fatty acids such as acetic, propionic and butyric acids are microbiota metabolites that can exert a series of physiological effects both in the intestine and other organs, including the central nervous system. The present work aimed to examine the effects of sodium acetate, propionate, and butyrate on the activity of large conductance Ca2+ activated K+ channels and calcium oscillations in rat pituitary GH3 cells. It has been shown that fatty acids under study cause a dose-dependent increase in the amplitude of total outward potassium currents and these effects are prevented by tetraethylammonium, a Ca2+ activated K+ channel blocker, indicating the involvement of Ca2+ activated K+ channels in the effects of fatty acids. It is worthy of note that fatty acids increased open probability of single channels with no changes in the amplitude and the mean channel open time. In addition, fatty acids were associated with a significant reduction in the amplitude and frequency of Ca2+ oscillations in GH3 cells. An increase in potassium conductance and a decrease in the intracellular Ca2+ level can mediate the effects of short-chain fatty acids in various excitable structures, such as a relaxation of intestinal and vascular smooth muscle cells, hyperpolarization of neurons, and the regulation of hormone and neurotransmitter release.

A Gating Mutation in Ryanodine Receptor Type 2 Rescues Phenotypes of Alzheimer's Disease Mouse Models by Upregulating Neuronal Autophagy.

It is well established that ryanodine receptors (RyanRs) are overactive in Alzheimer's disease (AD), and it has been suggested that inhibition of RyanR is potentially beneficial for AD treatment. In the present study, we explored a potential connection between basal RyanR activity and autophagy in neurons. Autophagy plays an important role in clearing damaged organelles and long-lived protein aggregates, and autophagy dysregulation occurs in both AD patients and AD animal models. Autophagy is known to be regulated by intracellular calcium (Ca2+) signals, and our results indicated that basal RyanR2 activity in hippocampal neurons inhibited autophagy through activation of calcineurin and the resulting inhibition of the AMPK (AMP-activated protein kinase)-ULK1 (unc-51-like autophagy-activating kinase 1) pathway. Thus, we hypothesized that increased basal RyanR2 activity in AD may lead to the inhibition of neuronal autophagy and accumulation of β-amyloid. To test this hypothesis, we took advantage of the RyanR2-E4872Q knock-in mouse model (EQ) in which basal RyanR2 activity is reduced because of shortened channel open time. We discovered that crossing EQ mice with the APPKI and APPPS1 mouse models of AD (both males and females) rescued amyloid accumulation and LTP impairment in these mice. Our results revealed that reduced basal activity of RyanR2-EQ channels disinhibited the autophagic pathway and led to increased amyloid clearance in these models. These data indicated a potential pathogenic outcome of RyanR2 overactivation in AD and also provided additional targets for therapeutic intervention in AD. Basal activity of ryanodine receptors controls neuronal autophagy and contributes to development of the AD phenotype.SIGNIFICANCE STATEMENT It is well established that neuronal autophagy is impaired in Alzheimer's disease (AD). Our results suggest that supranormal calcium (Ca2+) release from endoplasmic reticulum contributes to the inhibition of autophagy in AD and that reduction in basal activity of type 2 ryanodine receptors disinhibits the neuronal autophagic pathway and leads to increased amyloid clearance in AD models. Our findings directly link neuronal Ca2+ dysregulation with autophagy dysfunction in AD and point to additional targets for therapeutic intervention.

Late glacial retreat of the Lancaster Sound Ice Stream and early Holocene onset of Arctic/Atlantic throughflow in the Arctic Island channels

ABSTRACT Multiple proxies in three sediment cores from Northwestern Baffin Bay document the timing of Lancaster Sound Ice Stream (LSIS) retreat that led to Arctic–Atlantic throughflow in Parry Channel, an important source of freshwater that can impact the Atlantic Meridional Overturning Circulation. The Late Glacial to Holocene timing of ice retreat and channel opening and the responses of the regional ocean environment to these events are presented. We use quantitative mineral composition, foraminiferal assemblages, biogenic silica, ice-rafted debris (IRD), and 14C-based age models to document and date the events and environmental changes occurring during deglaciation of this major marine channel. Findings show that retreat of the LSIS into Lancaster Sound occurred before ~15.3 cal ka BP, about 800 years before the onset of major iceberg calving events from the LSIS, named the Baffin Bay Detrital Carbonate events (BBDC 1 and BBDC 0). The end of BBDC 0 occurred at ~10.6 cal ka BP, which coincides with the opening of Parry Channel. A marine environment productive of calcareous benthic and planktic foraminifera, with diminished meltwater, seasonal sea ice, warmer summer temperatures, and inflowing, nutrient-rich Arctic surface water characterizes the interval between the opening of Parry Channel and the opening of Nares Strait. Paired planktic and benthic 14C ages over this 2,200-year interval show diminishing age offsets suggesting progressive mixing of the upper ~850–900 m of the water column. The opening of Nares Strait by ~8.2 cal ka BP coincides with increased biogenic silica in the form of abundant, large centric diatoms and dissolution of CaCO3. The paucity of calcareous organisms after 8.2 cal ka BP resulted in poor chronological control in the cores to interpret changing environments after 8.2 cal ka BP.

Inward and outward currents of native and cloned K(ATP) channels (Kir6.2/SUR1) share single-channel kinetic properties

BackgroundThe ATP-sensitive K+ (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K+ channels, and the regulatory subunit sulfonylurea receptor (SURx). In insulin secreting β-cells in the endocrine part of the pancreas, where the channel is best studied, the K(ATP) channel consists of Kir6.2 and SUR1. Under physiological conditions, the K(ATP) channel current flow is outward at membrane potentials more positive than the K+ equilibrium potential around −80 mV. However, K(ATP) channel kinetics have been extensively investigated for inward currents and the single-channel kinetic model is based on this type of recording, whereas only a limited amount of work has focused on outward current kinetics. MethodsWe have estimated the kinetic properties of both native and cloned K(ATP) channels under varying ionic gradients and membrane potentials using the patch-clamp technique. ResultsAnalyses of outward currents in K(ATP) and cloned Kir6.2ΔC26 channels, alone or co-expressed with SUR1, show openings that are not grouped in bursts as seen for inward currents. Burst duration for inward current corresponds well to open time for outward current. ConclusionsOutward K(ATP) channel currents are not grouped in bursts regardless of membrane potential, and channel open time for outward currents corresponds to burst duration for inward currents.

Identification of a proton sensor that regulates conductance and open time of single hERG channels

The hERG potassium channel influences ventricular action potential duration. Extracellular acidosis occurs in pathological states including cardiac ischaemia. It reduces the amplitude of hERG current and speeds up deactivation, which can alter cardiac excitability. This study aimed to identify the site of action by which extracellular protons regulate the amplitude of macroscopic hERG current. Recordings of macroscopic and single hERG1a and 1b channel activity, mutagenesis, and the recent cryoEM structure for hERG were employed. Single hERG1a and 1b channels displayed open times that decreased with membrane depolarization, suggestive of a blocking mechanism that senses approximately 20% of the membrane electric field. This mechanism was sensitive to pH; extracellular acidosis reduced both hERG1a and1b channel open time and conductance. The effects of acidosis on macroscopic current amplitude and deactivation displayed different sensitivities to protons. Point mutation of a pair of residues (E575/H578) in the pore turret abolished the acidosis-induced decrease of current amplitude, without affecting the change in current deactivation. In single hERG1a channel recordings, the conductance of the double-mutant channel was unaffected by extracellular acidosis. These findings identify residues in the outer turret of the hERG channel that act as a proton sensor to regulate open time and channel conductance.

Mechanisms of GABAB receptor enhancement of extrasynaptic GABAA receptor currents in cerebellar granule cells

Many neurons, including cerebellar granule cells, exhibit a tonic GABA current mediated by extrasynaptic GABAA receptors. This current is a critical regulator of firing and the target of many clinically relevant compounds. Using a combination of patch clamp electrophysiology and photolytic uncaging of RuBi-GABA we show that GABAB receptors are tonically active and enhance extrasynaptic GABAA receptor currents in cerebellar granule cells. This enhancement is not associated with meaningful changes in GABAA receptor potency, mean channel open-time, open probability, or single-channel current. However, there was a significant (~40%) decrease in the number of channels participating in the GABA uncaging current and an increase in receptor desensitization. Furthermore, we find that adenylate cyclase, PKA, CaMKII, and release of Ca2+ from intracellular stores are necessary for modulation of GABAA receptors. Overall, this work reveals crosstalk between postsynaptic GABAA and GABAB receptors and identifies the signaling pathways and mechanisms involved.

Pathophysiology of and therapeutic options for a GABRA1 variant linked to epileptic encephalopathy

We report the identification of a de novo GABRA1 (R214C) variant in a child with epileptic encephalopathy (EE), describe its functional characterization and pathophysiology, and evaluate its potential therapeutic options. The GABRA1 (R214C) variant was identified using whole exome sequencing, and the pathogenic effect of this mutation was investigated by comparing wild-type (WT) α1 and R214C α1 GABAA receptor-expressing HEK cells. GABA-evoked currents in these cells were recorded using whole-cell, outside-out macro-patch and cell-attached single-channel patch-clamp recordings. Changes to surface and total protein expression levels of WT α1 and R214C α1 were quantified using surface biotinylation assay and western blotting, respectively. Finally, potential therapeutic options were explored by determining the effects of modulators, including diazepam, insulin, and verapamil, on channel gating and receptor trafficking of WT and R214C GABAA receptors. We found that the GABRA1 (R214C) variant decreased whole-cell GABA-evoked currents by reducing single channel open time and both surface and total GABAA receptor expression levels. The GABA-evoked currents in R214C GABAA receptors could only be partially restored with benzodiazepine (diazepam) and insulin. However, verapamil treatment for 24 h fully restored the function of R214C mutant receptors, primarily by increasing channel open time. We conclude that the GABRA1 (R214C) variant reduces channel activity and surface expression of mutant receptors, thereby contributing to the pathogenesis of genetic EE. The functional restoration by verapamil suggests that it is a potentially new therapeutic option for patients with the R214C variant and highlights the value of precision medicine in the treatment of genetic EEs.

Type I-like behavior of the type II α7 nicotinic acetylcholine receptor positive allosteric modulator A-867744.

Cognitive impairment often involves the decreased expression or hypofunction of alpha 7-type nicotinic acetylcholine receptors (α7 nAChRs). Agonists or positive allosteric modulators (PAMs) of α7 nAChRs are known to be potential treatments for dementias, different neurodegenerative disorders, pain syndromes and conditions involving inflammation. In some of these conditions, it is desirable to maintain the temporal precision of fast cholinergic events, while in others, this temporal precision is unnecessary. For this reason, the optimal therapeutic effect for distinct indications may require PAMs with different mechanisms of action. The two major mechanisms are called “type I”, which are compounds that augment α7 nAChR-mediated currents but maintain their characteristic fast kinetics; and “type II”, which are compounds that produce augmented and prolonged currents. In this study, we performed a kinetic analysis of two type II PAMs of the α7 nAChR: PNU-120596 and A-867744, using a fast perfusion method that allowed high temporal resolution. We characterized the type of modulation produced by the two compounds, the state-dependence of the modulatory action, and the interaction between the two compounds. We found fundamental differences between the modulation mechanisms by PNU-120596 and A-867744. Most importantly, during brief agonist pulses, A-867744 caused a strikingly type I-like modulation, while PNU-120596 caused a type II-like prolonged activation. Our results demonstrate that specific compounds, even though all labeled as type II PAMs, can behave in completely different ways, including their onset and offset kinetics, state preference, and single channel open time. Our results emphasize that subtle details of the mechanism of action may be significant in assessing the therapeutic applicability of α7 nAChR PAM compounds.

Ion Channel Properties of a Cation Channelrhodopsin, Gt_CCR4

We previously reported a cation channelrhodopsin, Gt_CCR4, which is one of the 44 types of microbial rhodopsins from a cryptophyte flagellate, Guillardia theta. Due to the modest homology of amino acid sequences with a chlorophyte channelrhodopsin such as Cr_ChR2 from Chlamydomonas reinhardtii, it has been proposed that a family of cryptophyte channelrhodopsin, including Gt_CCR4, has a distinct molecular mechanism for channel gating and ion permeation. In this study, we compared the photocurrent properties, cation selectivity and kinetics between well-known Cr_ChR2 and Gt_CCR4 by a conventional path clamp method. Large and stable light-induced cation conduction by Gt_CCR4 at the maximum absorbing wavelength (530 nm) was observed with only small inactivation (15%), whereas the photocurrent of Cr_ChR2 exhibited significant inactivation (50%) and desensitization. The light sensitivity of Gt_CCR4 was higher (EC50 = 0.13 mW/mm2) than that of Cr_ChR2 (EC50 = 0.80 mW/mm2) while the channel open life time (photocycle speed) was in the same range as that of Cr_ChR2 (25~30 ms for Gt_CCR4 and 10~15 ms for Cr_ChR2). This observation implies that Gt_CCR4 enables optical neuronal spiking with weak light in high temporal resolution when applied in neuroscience. Furthermore, we demonstrated high Na+ selectivity of Gt_CCR4 in which the selectivity ratio for Na+ was 37-fold larger than that for Cr_ChR2, which primarily conducts H+. On the other hand, Gt_CCR4 conducted almost no H+ and no Ca2+ under physiological conditions. These results suggest that ion selectivity in Gt_CCR4 is distinct from that in Cr_ChR2. In addition, a unique red-absorbing and stable intermediate in the photocycle was observed, indicating a photochromic property of Gt_CCR4.

Dual-Frequency Ultrasound Induces Neural Stem/Progenitor Cell Differentiation and Growth Factor Utilization by Enhancing Stable Cavitation.

Neural stem/progenitor cells (NSPCs) have the potential to serve as the basic materials for treating severe neural diseases and injuries. Ultrasound exposure is an effective therapy for nonunion fractures and healing fresh wounds through an easy and noninvasive application. According to the results of our preliminary study, low-intensity ultrasound (LIUS) promotes the attachment and differentiation of NSPCs. However, the parameters of and mechanisms by which LIUS induces NSPC differentiation remain unclear. To the best of our knowledge, no published studies have reported and compared the biological effects of dual-frequency and single-frequency LIUS on NSPCs. The purpose of this study is to systematically compare several LIUS parameters, including single-frequency, single-transducer dual-frequency ultrasound, burst, and continuous cycling stimulation at several intensities. Furthermore, synergistic effects of single-/dual-frequency LIUS combined with neural growth factor addition on NSPCs were also evaluated. Based on the results of the cytotoxicity assay, low-intensity (40 kPa) ultrasound does not damage NSPCs compared with that observed in the control group. The morphology and immunostaining results show that all experimental groups exposed to ultrasound exhibit neurite outgrowth and NSPC differentiation. In particular, dual-frequency ultrasound promotes NSPCs differentiation to a greater extent than single-frequency ultrasound. In addition, more complicated and denser neural networks are observed in the dual-frequency group. Neural growth factor addition increased the percentage of neurons formed, particularly in the groups stimulated with ultrasound. Among these groups, the dual-frequency group exhibited significant differences in the percentage of differentiated neurons compared with the single-frequency group. This study may the first to prove that dual-frequency LIUS exposure further enhances NSPC differentiation and the utilization of growth factors than single-frequency LIUS. Moreover, the result also revealed that dual-frequency ultrasound generated higher calcium ion influx and extended the channel opening time. A potential explanation is that dual-frequency ultrasound generates more stable cavitation than single-frequency LIUS, which may stimulate cell membrane mechanochannels and enhance calcium ion influx but does not damage them. This in vitro study may serve as a useful alternative for ultrasound therapy.

Competitive strategies and quality to counter parallel importation in global market

When selling products globally in geographically separated markets with price discrimination, manufacturers often have to compete with parallel importers (PIs) who sell “gray products,” purchasing the manufacturers’ authorized products in low-price markets and reselling them in high-price markets. In this paper, we show that manufacturers can use ex ante quality design along with distribution strategies to mitigate the negative impacts of parallel importation. We develop game-theoretic models for a supply chain with a manufacturer and a PI, in which the manufacturer needs to determine distribution structure (either selling through high-price market exclusively, or selling through two channels), product quality, and retail prices. If a dual-channel structure is selected, the manufacturer also should decide the channel opening time in the low-price market, either early or late. Our results suggest that (1) the manufacturer should strategically choose a lower quality level when designing its product to weaken the PI's competitiveness; (2) the manufacturer's optimal distribution strategy is determined by the gap between customers’ willingness to pay (WTP) in the two markets and the customer's tolerance for late consumption; and (3) parallel importation hurts the customer surplus (CS) and social welfare (SW) in the low-price market, and benefits CS and SW in the high-price market when the two markets are moderately balanced. We then consider several important extensions to provide additional insights. Firstly, we show that the appropriate marketing investment in the low market can effectively counter the PI. Secondly, we examine the impact of multiple competing PIs on the manufacturer's decisions, including distribution strategy, quality design, prices, and profit. Lastly, we find that the advertising effect may motivate the manufacturer to improve product quality and benefit both the manufacturer and PI if the effect is relatively strong.

P.016 A novel de novo GABRA1 mutation linked to epileptic encephalopathy: pathophysiology and potential therapeutic options

Background: Epileptic encephalopathy (EE) is a severe neurological disorder characterized by treatment-resistant seizures and poor neurodevelopmental outcomes. EE is associated with mutant genes, including those that encode for γ-aminobutyric acid type A (GABAA) receptor subunits. We identified a novel de novo GABRA1 mutation in a patient with EE, characterized its impact on GABAA receptor function, and sought potential therapeutic options. Methods: We described the clinical and electrophysiological features of a patient with a novel de novo GABRA1 (R214C) mutation; performed functional studies; and determined the effect of diazepam and insulin on wild type and mutant GABAA receptors. Results: The patient is a 10-year-old girl with EE, treatment-resistant seizures, intellectual disability and autism. Her GABRA1 (R214C) mutation dramatically decreased whole-cell GABA-evoked currents by reducing GABAA surface receptors, decreasing single channel open time, and altering channel kinetic properties. The combination of diazepam and insulin partially repaired these effects by enhancing channel activity and increasing the number of surface receptors, respectively. Conclusions: Diazepam and insulin partially mitigated a de novo GABRA1 (R214C) mutation’s effects on GABAA receptor number and function. Given the risks of insulin use, pharmacological agents with similar mechanisms of action but fewer side effects, such as IGF-1, should be studied and considered for clinical application.

Subunit composition critically determines pH sensitivity of epithelial sodium channels in Xenopus laevis

The Epithelial Sodium Channel (ENaC) mediates the selective flux of sodium ions across the apical membrane of epithelial cells. ENaC consists of three subunits (α,β,γ) and its activity at the cell surface is subject to a multitude of regulatory mechanisms, including proteolytic cleavage, extracellular pH and Na+ ions. Channel regulation may also depend on ENaC subunit composition, as an additional δ‐subunit can replace α‐ENaC in the heterotrimeric configuration. As the physiological implications of ENaC containing the δ‐subunit are poorly understood, we characterized the regulation of Xenopus δβγ‐ENaC by extracellular pH and Na+.Regulation of Xenopus δβγ‐ENaC by extracellular Na+ and pH was investigated by two‐electrode voltage‐clamp and cell‐attached Patch‐Clamp electrophysiology using the Xenopus oocyte expression system. Successive reduction of extracellular pH from pH 8 – 6 increased amiloride‐sensitive transmembrane currents of oocytes expressing δβγ‐ENaC by > 7 fold. In cell‐attached Patch‐Clamp recordings the pH of the pipette solution (pHpip = extracellular) affected δβγ‐ENaC open probability and kinetics. Compared to pHpip at 7.4, alkaline conditions (pHpip 8) decreased open probability as well as the mean channel open time, whereas under acidic conditions (pHpip 6) an increased open probability was accompanied by a reduction of average channel closed times. Transmembrane currents mediated by Xenopus δβγ‐ENaC are sensitive to sodium self‐inhibition, a phenomenon describing the fast inhibition of ENaC in the presence of high extracellular Na+ concentrations. Channel activation under acidic conditions (pH 7 & 6) was accompanied by a decrease in magnitude and apparent Na+‐affinity of self‐inhibition, whereas these effects were reversed under alkaline conditions (pH 8). Hypothesizing mutual regulation of δβγ‐ENaC activity by extracellular Na+ and pH, we examined whether neutralization of pH‐sensitive aspartates in the δ‐subunit affected channel self‐inhibition and regulation by pH. Specific aspartates in a subunit domain corresponding to a putative Na+‐sensing site of mouse α‐ENaC (Kashlan et al., 2015, J Biol Chem 290:568–76) were replaced by asparagines via site‐directed mutagenesis (δD293N, δD296N, δD293,6N). All mutations decreased pH‐mediated channel activation. Notably, ENaC containing the δD296N mutation also displayed a decreased sodium self‐inhibition, which was not observed in channels bearing a single δD293N mutation. Furthermore, human δβγ‐ENaCs which have a reduced sodium self‐inhibition were not activated by acidic pH as observed with the Xenopus orthologue displaying a strong sodium self‐inhibition.In conclusion, these results demonstrate that activity of ENaC containing the δ‐subunit in Xenopus laevis is sensitive to changes in the extracellular pH, whereas ENaC containing the α‐subunit is not. Proton‐mediated activation of ENaC involves changes in single channel kinetics and a reduction of sodium self‐inhibition. Thus, sensitivity of this amphibian ENaC to extracellular pH is critically determined by its subunit composition.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

De novo BK channel variant causes epilepsy by affecting voltage gating but not Ca2+ sensitivity.

Epilepsy is one of the most common neurological diseases and it causes profound morbidity and mortality. We identified the first de novo variant in KCNMA1 (c.2984 A > G (p.(N995S)))-encoding the BK channel-that causes epilepsy, but not paroxysmal dyskinesia, in two independent families. The c.2984 A > G (p.(N995S)) variant markedly increased the macroscopic potassium current by increasing both the channel open probability and channel open dwell time. The c.2984 A > G (p.(N995S)) variant did not affect the calcium sensitivity of the channel. We also identified three other variants of unknown significance (c.1554 G > T (p.(K518N)), c.1967A > C (p.(E656A)), and c.3476 A > G (p.(N1159S))) in three separate patients with divergent epileptic phenotypes. However, these variants did not affect the BK potassium current, and are therefore unlikely to be disease-causing. These results demonstrate that BK channel variants can cause epilepsy without paroxysmal dyskinesia. The underlying molecular mechanism can be increased activation of the BK channel by increased sensitivity to the voltage-dependent activation without affecting the sensitivity to the calcium-dependent activation. Our data suggest that the BK channel may represent a drug target for the treatment of epilepsy. Our data highlight the importance of functional electrophysiological studies of BK channel variants in distinguishing whether a genomic variant of unknown significance is a disease-causing variant or a benign variant.

Counteracting desensitization of human α7-nicotinic acetylcholine receptors with bispyridinium compounds as an approach against organophosphorus poisoning

Irreversible inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and overstimulation of muscarinic and nicotinic receptors accounts for the acute toxicity of organophosphorus compounds (OP). Accordingly, the mainstay pharmacotherapy against poisoning by OP comprises the competitive muscarinic acetylcholine receptor antagonist atropine to treat muscarinic effects and, in addition, oximes to reactivate inhibited AChE. A therapeutic gap still remains in the treatment of desensitized nicotinic acetylcholine receptors following OP exposure. Hereby, nicotinic effects result in paralysis of the central and peripheral respiratory system if untreated. Thus, these receptors pose an essential target for therapeutic indication to address these life-threatening nicotinic symptoms of the cholinergic crisis. Identification of ligands regulating dynamic transitions between functional states by binding to modulatory sites appears to be a promising strategy for therapeutic intervention. In this patch clamp study, the ability of differently substituted bispyridinium non-oximes to “resensitize” i.e. to recover the activity of desensitized human homomeric α7-type nAChRs stably transfected in CHO cells was investigated and compared to the already described α7-specific positive allosteric modulator PNU-120596. The structures of these bispyridinium analogues were based on the lead structure of the tert-butyl-substituted bispyridinium propane MB327, which has been shown to have a positive therapeutic effect due to a non-competitive antagonistic action at muscle-type nAChRs in vivo and has been found to have a positive allosteric activity at neuronal receptors in vitro. Prior to test compounds, desensitization of hα7-nAChRs was verified by applying an excess of nicotine revealing activation at low, and desensitization at high concentrations. Thereby, desensitization could be reduced by modulation with PNU-120596. Desensitization was further verified by dose-response profiles of agonists, carbamoylcholine and epibatidine in the absence and presence of PNU-120596. Although less pronounced than PNU-120596 and the lead structure MB327, bispyridinium compounds, particularly those substituted at position 3 and 4, resensitized the nicotine desensitized hα7-nAChRs in a concentration-dependent manner and prolonged the mean channel open time. In summary, identification of more potent compounds able to restore nAChR function in OP intoxication is needed for development of a putative efficient antidote.

Single Channel Analysis of Isoflurane and Ethanol Enhancement of Taurine-Activated Glycine Receptors.

The amino acid taurine is an endogenous ligand acting on glycine receptors (GlyRs), which is released by astrocytes in many brain regions, such as the nucleus accumbens and prefrontal cortex. Taurine is a partial agonist with an efficacy significantly lower than that of glycine. Allosteric modulators such as ethanol and isoflurane produce leftward shifts of glycine concentration-response curves but have no effects at saturating glycine concentrations. In contrast, in whole-cell electrophysiology studies these modulators increase the effects of saturating taurine concentrations. A number of possible mechanisms may explain these enhancing effects, including modulator effects on conductance, channel open times, or channel closed times. We used outside-out patch-clamp single channel electrophysiology to investigate the mechanism of action of 200 mM ethanol and 0.55 mM isoflurane in enhancing the effects of a saturating concentration of taurine. Neither modulator enhanced taurine-mediated conductance. Isoflurane increased the probability of channel opening. Isoflurane also increased the lifetimes of the two shortest open dwell times while both agents decreased the likelihood of occurrence of the longest-lived intracluster channel-closing events. The mechanism of enhancement of GlyR functioning by these modulators is dependent on the efficacy of the agonist activating the receptor and the concentration of agonist tested.

Current-direction/amplitude-dependent single channel gating kinetics of mouse pannexin 1 channel: a new concept for gating kinetics

The detailed single-channel gating kinetics of mouse pannexin 1 (mPanx1) remains unknown, although mPanx1 is reported to be a voltage-activated anion-selective channel. We investigated characteristics of single-channel conductances and opening and closing rates of mPanx1 using patch-clamp techniques. The unitary current of mPanx1 shows outward rectification with single-channel conductances of ~20 pS for inward currents and ~80 pS for outward currents. The channel open time for outward currents (Cl− influx) increases linearly as the amplitude of single channel currents increases, while the open time for inward currents (Cl− efflux) is constant irrespective of changes in the current amplitude, as if the direction and amplitude of the unitary current regulates the open time. This is supported by further observations that replacement of extracellular Cl− with gluconate− diminishes the inward tail current (Cl− efflux) at a membrane potential of −100 mV due to the lowered outward current (gluconate− influx) at membrane potential of 100 mV. These results suggest that the direction and rate of charge-carrier movement regulate the open time of mPanx1, and that the previously reported voltage-dependence of Panx1 channel gating is not directly mediated by the membrane potential but rather by the direction and amplitude of currents through the channel.

Correction to 'Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels'.

Open AccessMoreSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail Cite this article Proks Peter, Puljung Michael C., Vedovato Natascia, Sachse Gregor, Mulvaney Rachel and Ashcroft Frances M. 2016Correction to ‘Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels’Phil. Trans. R. Soc. B3712016033020160330http://doi.org/10.1098/rstb.2016.0330SectionOpen AccessCorrectionCorrection to ‘Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels’ Peter Proks Peter Proks Google Scholar Find this author on PubMed Search for more papers by this author , Michael C. Puljung Michael C. Puljung Google Scholar Find this author on PubMed Search for more papers by this author , Natascia Vedovato Natascia Vedovato Google Scholar Find this author on PubMed Search for more papers by this author , Gregor Sachse Gregor Sachse Google Scholar Find this author on PubMed Search for more papers by this author , Rachel Mulvaney Rachel Mulvaney Google Scholar Find this author on PubMed Search for more papers by this author and Frances M. Ashcroft Frances M. Ashcroft Google Scholar Find this author on PubMed Search for more papers by this author Peter Proks Peter Proks Google Scholar Find this author on PubMed Search for more papers by this author , Michael C. Puljung Michael C. Puljung Google Scholar Find this author on PubMed Search for more papers by this author , Natascia Vedovato Natascia Vedovato Google Scholar Find this author on PubMed Search for more papers by this author , Gregor Sachse Gregor Sachse Google Scholar Find this author on PubMed Search for more papers by this author , Rachel Mulvaney Rachel Mulvaney Google Scholar Find this author on PubMed Search for more papers by this author and Frances M. Ashcroft Frances M. Ashcroft Google Scholar Find this author on PubMed Search for more papers by this author Published:05 September 2016https://doi.org/10.1098/rstb.2016.0330This article corrects the followingReview ArticleRunning out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channelshttps://doi.org/10.1098/rstb.2015.0426 Peter Proks, Michael C. Puljung, Natascia Vedovato, Gregor Sachse, Rachel Mulvaney and Frances M. Ashcroft volume 371issue 1700Philosophical Transactions of the Royal Society B: Biological Sciences05 August 2016Phil. Trans. R. Soc. B371, 20150426 (2016; Published 4 July 2016) (doi:10.1098/rstb.2015.0426)The x-axis labels in figure 2b are incorrect. The corrected figure 2 is given below. Figure 2. Effect of rundown on single-channel KATP channel properties. (a) Representative 1 min recording of single Kir6.2-G334D/SUR1 channels at −60 mV in the cell-attached configuration (a, Po = 0.83) and after 5-min after patch excision (b, Po = 0.22). (b) Distributions of channel open times (i), closed times (ii) and burst duration (iii) before (pale grey bars) and after (dark grey bars) rundown. The distributions were fitted with probability density functions that gave the following values for the individual components. Before rundown: mean open time, 2.7 ms; three apparent closed states with mean values of 0.3 ms (98.5%), 2 ms (0.8%) and 12 ms (0.7%); and a single burst state with a mean duration of 120 ms. After rundown: mean open time 2.2 ms; five apparent closed states with mean values 0.3 ms (88.9%), 3 ms (3.3%), 19 ms (4.3%), 82 ms (3.1%) and 650 ms (0.4%); and three apparent burst states with mean durations of 23 ms (66%), 5 ms (20.5%) and 0.5 ms (13.5%). Vertical lines indicate the mean values for open times, short closed times and the major burst duration component, and illustrate that rundown has no effect on the intraburst closed times, but reduces the mean open time and burst duration. The methods and solutions used are as described in [6].Download figureOpen in new tabDownload PowerPoint Previous Article VIEW FULL TEXT DOWNLOAD PDF FiguresRelatedReferencesDetailsRelated articlesRunning out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels05 August 2016Philosophical Transactions of the Royal Society B: Biological Sciences This Issue05 September 2016Volume 371Issue 1702Theme issue ‘From DNA barcodes to biomes’ compiled and edited by Paul D. N. Hebert, Mehrdad Hajibabaei and Peter M. Hollingsworth Article InformationDOI:https://doi.org/10.1098/rstb.2016.0330PubMed:27481794Published by:Royal SocietyPrint ISSN:0962-8436Online ISSN:1471-2970History: Published online05/09/2016Published in print05/09/2016 License:© 2016 The Authors.Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Citations and impact Subjectsbiophysicsphysiology

Functional and structural interactions between selective serotonin reuptake inhibitors and nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in the regulation of mood and their related disorders such as anxiety and major depressive disorder. For example, there is a clear and strong association between major depression and smoking, where depressed patients smoke twice more than the non-depressed population, and opposite to that, nicotine consumption may lead to depressive symptoms. In patients with major depressive disorder, although the number of nAChRs is not altered, there is a low availability of nAChRs, possibly caused by higher endogenous ACh levels and the consequent nAChR desensitization, consistent with the cholinergic hypothesis of depression, in which the cholinergic systems is hypersensitive. Moreover, studies using animal models of depression reveal that nicotine-evoked serotonin release is mediated by nAChR activation compensating the deficiency of serotonin in depressed conditions. It was also reported that several common clinically prescribed antidepressants, including selective serotonin reuptake inhibitors (SSRIs), inhibit different nAChRs by non-competitive mechanisms, including ion channel blockade and accelerating nAChR desensitization. More specifically, SSRIs decrease channel open time without changing the single-channel conductance, indicating that these antidepressants act as open-channel blockers. Furthermore, inactive doses of citalopram, a SSRI antidepressant, and of PNU-28298, an α7 nAChR agonist, exert antidepressant-like effects. According with functional and structural results, SSRIs (e.g., fluoxetine) and tricyclic antidepressants (e.g., imipramine) bind to overlapping sites located within the nAChR ion channel in the desensitized and resting states.