Block Of Currents Research Articles

Electrophysiology of Hypokalemia and Hyperkalemia.

Electrophysiology of Hypokalemia and Hyperkalemia.

Characterization and structure-activity relationship of natural flavonoids as hERG K+ channel modulators

ObjectivesFlavonoids are present in varying concentrations in plant foods and have been reported to have numerous pharmacological activities, such as anti-cancer, antioxidant, anti-inflammatory, hepatoprotective, and vasodilator effects. We found that quercetin, fisetin, and some related flavonoid derivatives could inhibit human ether-à-go-go-related gene (hERG) K+ channels. Key findingsIn this study, we tested the effects of a series of flavonoids on the hERG K+ channel expressed in HEK293 cells. For the first time, we demonstrate that quercetin and fisetin (Fise) are potent hERG current blockers. The 50% inhibiting concentration (IC50) and maximum efficacy (Emax) of quercetin were 11.8±0.9μM and 82±2%, while those of fisetin were 38.4±6μM and 100±6%, respectively. Luteolin (Lute) was a less potent inhibitor of hERG current (48±1% at 100μM). Galangin, kaempferol, and isorhamnetin (100μM) showed weaker activity on the hERG currents. ConclusionThese results suggest that quercetin, fisetin, and luteolin are potent hERG K+ channel inhibitors and reveal the structure-activity relationship of natural flavonoids.

Translocation and Binding in the Recognition of Short Oligonucleotides by a Biological Nanopore

Polymer interactions with pore-forming membrane proteins, as evidenced by the resulting block of ionic current, may be classified into two types: (1) the threading of an extended polymer chain of which only a small length contributes to the block and (2) a binding reaction, where the polymer enters the pore entirely and interacts as a particle. The former phenomenon typically occurs in dilute aqueous electrolyte solutions (e.g. KCl <1 M) and requires long and charged molecules and can allow sequencing (e.g. of DNA). In contrast, the binding reaction typically occurs with short, neutral polymer molecules, requires high salinity (e.g. KCl 3-4 M) and enables the high-resolution discrimination of polymer masses1. The aerolysin nanopore has recently been shown to strongly interact with short adenine oligonucleotides (A3-A10) and this interaction allows mass discrimination on the basis of the depth of block of ionic current induced by the binding of the analyte,2 suggesting a binding-type interaction. Using low-noise current recording, we have indentified strong dynamics of this interaction between DC and 50 kHz and indentified short visits to deeper blocked states preceding and following the principal, mass-dependent state (pre- and post-blocks). Statistical analysis indicates that the probability of post- but not pre-blocks decreases with (1) oligomer length and (2) transmembrane voltage. We interpret this finding in terms of a combined translocation and binding interaction, probably involving several binding sites for DNA in the pore. In line with this hypothesis, longer polynucleotides (A30) showed stable and long-lasting interactions with near-complete block of current.(1) Robertson et al. Proc. Natl. Acad. Sci. U S A 2007, 104, 8207-8211.(2) Cao et al. Nature Nanotechnology 2016, 1-7.

Probing the Movement of the Ball and Chain during N-type Inactivation in Kv Channels

N-type inactivation is a mechanism in certain Kv channels where the N-terminal peptide occludes the pore upon depolarization resulting in block of ionic currents (ball-and-chain). Numerous mutational studies have characterized N-type inactivation functionally, while X-ray crystal structures have yet to include the ball-and-chain structure. It still remains unknown how far the N-terminus travels during N-type inactivation and where it is located in the resting state. Does it reside in a fixed position near the T1 window or is it randomly floating in the cytosol? By incorporating a fluorescent unnatural amino acid (Anap) into the N-terminus and into receptor sites (T1 window and S4-S5 linker) we directly tracked the movement of the ball peptide using voltage clamp fluorometry in Xenopus oocytes. Voltage dependency and kinetics of ball-peptide movement correlated with N-type inactivating currents. Furthermore, we address the question as to whether the fluorescence changes are caused by polarity changes or quenching, by measuring the Anap emission spectra at both resting and activated state.

Characteristics of Ivabradine-Sensitive Currents in Mouse Sinoatrial Node Myocytes

Cardiac pacemaking is driven by spontaneous action potentials (APs) in sinoatrial node myocytes (SAMs). The funny current (If) is thought play a role in the generation of pacemaker activity in SAMs, but the degree to which it contributes is incompletely understood. In this study, we used AP clamp experiments and the If blocker, ivabradine, to investigate the role of If in pacemaking. Spontaneous APs were recorded from acutely-isolated SAMs from mice, and ∼10 s of the AP recording was incorporated into a voltage clamp command. This AP clamp protocol elicited both inward and outward ivabradine-sensitive currents in SAMs. If was defined as the ivabradine-sensitive current remaining in the presence of a pharmacological cocktail of sodium, calcium and potassium channel blockers. Measurable inward ivabradine-sensitive current was evident during diastole in 57% of all cells and was generally larger in cells with the most negative diastolic voltages. Although we found that 30 μM ivabradine also blocked both L- and T-type calcium currents in SAMs, the majority of the ivabradine-sensitive inward current in the AP clamp experiments appeared to be If because control experiments showed that the drug cocktail blocked ∼85% of the calcium currents. Ivabradine also blocked a substantial outward current during the AP upstroke and repolarization phases. This outward ivabradine-sensitive current likely includes a contribution of If, which results from its relatively slow deactivation. However some of the outward ivabradine-sensitive current may also reflect off-target block of potassium currents by ivabradine. We are currently assaying the contribution of potassium currents to the outward ivabradine-sensitive current. Our results suggest that If is active not only during diastole, but also throughout the AP upstroke and repolarization phases of the mouse SAM AP.

Simulation of ventricular rate control during atrial fibrillation using ionic channel blockers

BackgroundThe atrioventricular (AV) node is the only compartment that conducts an electrical impulse between the atria and the ventricles. The main role of the AV node is to facilitate efficient pumping by conducting excitation slowly between the two chambers as well as reduce the ventricular rate during atrial fibrillation (AF). MethodsUsing computer simulations, we investigated excitation conduction from the right atrium to the bundle of His during high-rate atrial excitation with or without partial blocking of the calcium or potassium ionic current. ResultsOur simulations revealed differences in rate reduction and repolarization effects between calcium and potassium current blocking and high degree of potassium current blocking required to reduce the ventricular rate during AF. ConclusionsOur simulation results explain why potassium current blockers are not recommended for controlling ventricular rate during AF.

Heart failure modulates electropharmacological characteristics of sinoatrial nodes.

The impact of heart failure (HF) on sinoatrial node (SAN) channel regulation and electropharmacological responses has remained elusive. The present study aimed to investigate the effects of HF on the electrical activity of SANs with and without pharmacological interventions. Action potentials (APs) were recorded in isolated SANs from normal rabbits (control) and those with HF (rapid ventricular pacing for 4 weeks) prior to and after administration of a funny current blocker (ivabradine; 0.1, 0.3, 3 or 10 µM), a calmodulin kinase II inhibitor (KN-93; 0.3 or 3 µM), a sarcoplasmic reticulum Ca2+ release inhibitor (ryanodine; 0.3 or 3 µM), a sodium current inhibitor (tetrodotoxin; 1, 3 or 10 µM) and a late sodium current inhibitor (ranolazine; 10 µM). Western blot analysis was used to investigate the protein expression in SANs from normal rabbits and those with HF. Control SANs had a higher beating rate than SANs from rabbits with HF (2.3±0.1 vs. 1.5±0.1 Hz; P<0.001). Similarly, ivabradine (10 µM), KN-93 (3 µM), ranolazine (10 µM) and ryanodine (3 µM) decreased the beating rates of SANs in the control (n=6) and HF (n=6) groups. Ivabradine treatment resulted in a higher incidence of AP block in HF vs. control SANs (66.7 vs. 0%; P<0.05). Tetrodotoxin (1, 3 or 10 µM) decreased the beating rate to a higher extent in SANs from rabbits with HF than in those from control rabbits and induced a higher incidence of AP block (66.7 vs. 0%; P<0.05). Furthermore, SANs from rabbits with HF had higher protein levels of phospholamban (PLB) and lower levels of hyperpolarization-activated cyclic nucleotide-gated potassium channel 4, ryanodine receptor and phosphorylated PLB than control SANs. In conclusion, HF modulates electropharmacological responses in the SAN by channel regulation, which may result in SAN dysfunction.

Angina and Its Management.

Angina pectoris is defined as substernal chest pain, pressure, or discomfort that is typically exacerbated by exertion and/or emotional stress, lasts greater than 30 to 60 seconds, and is relieved by rest and nitroglycerin. There are approximately 10 million people in the United States who have angina, and there are over 500 000 cases diagnosed per year. Several studies now show that angina itself is a predictor of major adverse cardiac events. In addition, angina is a serious morbidity that impedes quality of life and should be treated. In the United States, pharmacologic therapy for angina includes β-blockers, nitrates, calcium channel blockers, and the late sodium current blocker ranolazine. In other countries, additional pharmacologic agents include trimetazidine, ivabradine, nicorandil, fasudil, and others. Revascularization is indicated in certain high-risk individuals and also has been shown to improve angina. However, even after revascularization, a substantial percentage of patients return with recurrent or continued angina, requiring newer and better therapies. Treatment for refractory angina not amenable to usual pharmacologic therapies or revascularization procedures, includes enhanced external counterpulsation, transmyocardial revascularization, and stem cell therapy. Angina continues to be a significant cause of morbidity. Therapy should be geared not only to treating the risk factors for atherosclerotic disease and improving survival but should also be aimed at eliminating or reducing the occurrence of angina and improving the ability of patients to be active.

The delayed strengthening of synaptic connectivity in the amygdala depends on NMDA receptor activation during acute stress.

There is growing evidence that stress leads to contrasting patterns of structural plasticity in the hippocampus and amygdala, two brain areas implicated in the cognitive and affective symptoms of stress‐related psychiatric disorders. Acute stress has been shown to trigger a delayed increase in the density of dendritic spines in the basolateral amygdala (BLA) of rodents. However, the physiological correlates of this delayed spinogenesis in the BLA remain unexplored. Furthermore, NMDA receptors (NMDARs) have been known to underlie chronic stress‐induced structural plasticity in the hippocampus, but nothing is known about the role of these receptors in the delayed spinogenesis, and its physiological consequences, in the BLA following acute stress. Here, using whole‐cell recordings in rat brain slices, we find that a single exposure to 2‐h immobilization stress enhances the frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs) recorded from principal neurons in the BLA 10 days later. This was also accompanied by faster use‐dependent block of NMDA receptor currents during repeated stimulation of thalamic inputs to the BLA, which is indicative of higher presynaptic release probability at these inputs 10 days later. Furthermore, targeted in vivo infusion of the NMDAR‐antagonist APV into the BLA during the acute stress prevents the increase in mEPSC frequency and spine density 10 days later. Together, these results identify a role for NMDARs during acute stress in both the physiological and morphological strengthening of synaptic connectivity in the BLA in a delayed fashion. These findings also raise the possibility that activation of NMDA receptors during stress may serve as a common molecular mechanism despite the divergent patterns of plasticity that eventually emerge after stress in the amygdala and hippocampus.

Differential Effects of Toluene and Ethanol on Dopaminergic Neurons of the Ventral Tegmental Area.

Drugs of abuse increase the activity of dopaminergic neurons of the ventral tegmental area (VTA), and output from the VTA is critical for both natural and drug-induced reward and reinforcement. Ethanol and the abused inhalant toluene both enhance VTA neuronal firing, but the mechanisms of this effect is not fully known. In this study, we used extracellular recordings to compare the actions of toluene and ethanol on DA VTA neurons. Both ethanol and toluene increased the firing rate of DA neurons, although toluene was ~100 times more potent than ethanol. The mixed ion channel blocker quinine (100 μM) blocked the increases in firing produced by ethanol and toluene, indicating some similarity in mechanisms of excitation. A mixture of antagonists of GABA and cholinergic receptors did not prevent toluene-induced or ethanol-induced excitation, and toluene-induced excitation was not altered by co-administration of ethanol, suggesting independent mechanisms of excitation for ethanol and toluene. Concurrent blockade of NMDA, AMPA, and metabotropic glutamate receptors enhanced the excitatory effect of toluene while having no significant effect on ethanol excitation. Nicotine increased firing of DA VTA neurons, and this was blocked by the nicotinic antagonist mecamylamine (1 μM). Mecamylamine did not alter ethanol or toluene excitation of firing but the muscarinic antagonist atropine (5 μM) or a combination of GABA antagonists (bicuculline and CGP35348, 10 μM each) reduced toluene-induced excitation without affecting ethanol excitation. The Ih current blocker ZD7288 abolished the excitatory effect of toluene but unlike the block of ethanol excitation, the effect of ZD7288 was not reversed by the GIRK channel blocker barium, but was reversed by GABA antagonists. These results demonstrate that the excitatory effects of ethanol and toluene have some similarity, such as block by quinine and ZD7288, but also indicate that there are important differences between these two drugs in their modulation by glutamatergic, cholinergic, and GABAergic receptors. These findings provide important information regarding the actions of abused inhalants on central reward pathways, and suggest that regulation of the activation of central dopamine pathways by ethanol and toluene partially overlap.

Activation of MEK/ERK signaling contributes to the PACAP-induced increase in guinea pig cardiac neuron excitability.

Pituitary adenylate cyclase (PAC)-activating polypeptide (PACAP) peptides (Adcyap1) signaling at the selective PAC1 receptor (Adcyap1r1) participate in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, and as these neurons are readily accessible, this neuronal system is particularly amenable to study of PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated phosphorylated ERK and, in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels (Ih) and another flowing through low-voltage-activated calcium channels (IT), which support the peptide-induced increase in excitability. Thus a PACAP- and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulates neuronal excitability. Despite prior work implicating PACAP downregulation of the KV4.2 potassium channel in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target, the NaV1.7 sodium channel, and treatment with the selective NaV1.7 channel inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate the NaV1.7 channel, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.

Abstract 384: Evaluating Late Sodium Current in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Background: Inhibiting late sodium current (I NaL ) reduced drug-induced QTc prolongation in a recent clinical trial. Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have emerged as a valuable tool in preclinical assessment of multichannel blocking drugs’ potential to prolong QT and induce arrhythmias. However, sodium channels in commercially available iPSC-CMs are known to be under expressed, necessitating investigation into the presence and effects of I NaL in this electrophysiological model. Methods and Results: A platform combining simultaneous measurements of field potential and contraction (xCELLigence RTCA CardioECR, ACEA Biosciences) was used to assess the acute effects of three I NaL enhancing drugs, ATX-II, ibutilide, and alfuzosin, given alone or in combination with an I NaL blocker, lidocaine in iPSC-CMs (iCell Cardiomyocytes 2 , Cellular Dynamics). Additionally, dofetilide, diltiazem, and lidocaine alone were included as positive controls for hERG, L-type calcium, and sodium channel block. ATX-II, a potent and specific I NaL enhancer, caused significant dose dependent rate-corrected field potential duration (FPDc) prolongation, which was then subsequently reduced in a dose dependent manner by the addition of lidocaine. At 100 nM ATX-II prolonged the FPDc by 1153.8 ± 135.8 ms from 360.5 ± 16.4 ms at the baseline, which was then reduced to 537 ± 37.4 ms with the addition of 30 μM lidocaine. Ibutilide (0.1-1 μM), a class III antiarrhythmic, caused beating rate decreases and early after depolarizations (EADs) that were not affected by lidocaine addition. Alfuzosin, which increases both peak and late sodium currents, caused dose-dependent reduction of beating rate, FPDc prolongation, and EADs at 5 μM and 10 μM. Alfuzosin-induced EADs were mitigated by addition of lidocaine (5-15 μM). Conclusions: Late sodium current enhancers prolonged repolarization and induced arrhythmias in human iPSC-CMs. These effects were reversed by addition of lidocaine, a specific late sodium current blocker. These results are consistent with the late sodium current being present in iPSC-CMs in the presence of a late sodium current enhancer, which may have implications for drug safety testing.

The Significance of the Late Na+ Current for Arrhythmia Induction and the Therapeutic Antiarrhythmic Potential of Ranolazine.

The purpose of this article is to review the basis of arrhythmogenesis, the functional and clinical role of the late Na current, and its therapeutic inhibition. Under pathological conditions such as ischemia and heart failure this current is abnormally enhanced and influences cellular electrophysiology as a proarrhythmic substrate in myocardial pathology. Ranolazine the only approved late Na current blocker has been demonstrated to produce antiarrhythmic effects in the atria and the ventricle. We summarize recent experimental and clinical studies of ranolazine and other experimental late Na current blockers and discuss the significance of the available data.

Heterosynaptic modulation of evoked synaptic potentials in layer II of the entorhinal cortex by activation of the parasubiculum.

The superficial layers of the entorhinal cortex receive sensory and associational cortical inputs and provide the hippocampus with the majority of its cortical sensory input. The parasubiculum, which receives input from multiple hippocampal subfields, sends its single major output projection to layer II of the entorhinal cortex, suggesting that it may modulate processing of synaptic inputs to the entorhinal cortex. Indeed, stimulation of the parasubiculum can enhance entorhinal responses to synaptic input from the piriform cortex in vivo. Theta EEG activity contributes to spatial and mnemonic processes in this region, and the current study assessed how stimulation of the parasubiculum with either single pulses or short, five-pulse, theta-frequency trains may modulate synaptic responses in layer II entorhinal stellate neurons evoked by stimulation of layer I afferents in vitro. Parasubicular stimulation pulses or trains suppressed responses to layer I stimulation at intervals of 5 ms, and parasubicular stimulation trains facilitated layer I responses at a train-pulse interval of 25 ms. This suggests that firing of parasubicular neurons during theta activity may heterosynaptically enhance incoming sensory inputs to the entorhinal cortex. Bath application of the hyperpolarization-activated cation current (Ih) blocker ZD7288 enhanced the facilitation effect, suggesting that cholinergic inhibition of Ih may contribute. In addition, repetitive pairing of parasubicular trains and layer I stimulation induced a lasting depression of entorhinal responses to layer I stimulation. These findings provide evidence that theta activity in the parasubiculum may promote heterosynaptic modulation effects that may alter sensory processing in the entorhinal cortex.

Islanding detection method based on a new approach to voltage phase angle of constant power inverters

This study presents a new islanding detection approach using the notion of active methods. A difference between the instantaneous and nominal voltage phase angle (VPA) of a distributed generation (DG) is applied to inverter-based interface control. After the islanding phenomenon, currents id and iq will be changed by adding the variation of the point of common coupling VPA to input of d–q transformation block of currents. This subsequently causes the voltage and frequency deviations using for detecting island. Active islanding detection techniques usually leave adverse effects on power quality. The considerable advantages of the proposed method include insignificant negative effects on the power quality in the normal operation of the power system. The performance of the proposed islanding detection method is evaluated by some common test presented in UL1741; in addition, the results of some tests are presented for multiple DGs that show the proposed method does not cause significant interference between two DGs in the operational mode. Analogous to the case of a single DG, the island was also detected well in the case of multiple DGs.

Sodium-calcium exchanger and R-type Ca2+ channels mediate spontaneous [Ca2+]i oscillations in magnocellular neurones of the rat supraoptic nucleus

Isolated supraoptic neurones generate spontaneous [Ca2+]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca2+, Na+), Na+/Ca2+ exchanger (NCX), intracellular Ca2+ release channels (InsP3Rs and RyRs), Ca2+ storage organelles, plasma membrane Ca2+ pump and intracellular signal transduction cascades into spontaneous Ca2+ activity. While removal of extracellular Ca2+ or incubation with non-specific voltage-gated Ca2+ channel (VGCC) blocker Cd2+ suppressed the oscillations, neither Ni2+ nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca2+]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca2+]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na+ or application of an inhibitor of the reverse operation mode of Na+/Ca2+ exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca2+]i activity. Exposure of neurones to Ca2+ mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K+ current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca2+ channels and the NCX fluctuating between forward and reverse modes.

Fast-onset lidocaine block of rat NaV1.4 channels suggests involvement of a second high-affinity open state

Local anesthetics (LAs) block resting, open, and inactivated states of voltage-gated Na+ channels where inactivated states are thought to bind with highest affinity. However, reports of fast-onset block occurring over milliseconds hint at high-affinity block of open channels. Movement of voltage-sensor domain IV-segment 4 (DIVS4) has been associated with high affinity LA block termed voltage-sensor block (VSB) that also leads to a second open state. These observations point to a second high-affinity open state that may underlie fast-onset block. To test for this state, we analyzed the modulation of Na+ currents by lidocaine and its quaternary derivative (QX222) from heterologously expressed (Xenopus laevis oocytes) rat skeletal muscle μ1 NaV1.4 (rSkM1) with β1 (WT-β1), and a mutant form (IFM-QQQ mutation in the III–IV interdomain, QQQ) lacking fast inactivation, in combination with Markov kinetic gating models. 100μM lidocaine induced fast-onset (τonset≈2ms), long-lived (τrecovery≈120ms) block of WT-β1 macroscopic currents. Lidocaine blocked single-channel and macroscopic QQQ currents in agreement with our previously described mechanism of dual, open-channel block (DOB mechanism). A DOB kinetic model reproduced lidocaine effects on QQQ currents. The DOB model was extended to include trapping fast-inactivation and activation gates, and a second open state (OS2); the latter arising from DIVS4 translocation that precedes inactivation and exhibits high-affinity, lidocaine binding (apparent Kd=25μM) that accords with VSB (DOB-S2VSB mechanism). The DOB-S2VSB kinetic model predicted fast-onset block of WT-β1. The findings support the involvement of a second, high-affinity, open state in lidocaine modulation of Na+ channels.

An experimental study to determine the obstacle height required for the control of subcritical and supercritical gravity currents

Turbidity currents are important phenomena involved in sediment processes in long and deep reservoirs. Deviation or dissipation of the turbidity currents in reservoirs can be achieved by using obstacles. In this paper, we study the effects of obstacle height on complete control of both subcritical and supercritical gravity currents (.7 ≤ Frd ≤ 1.37) with various inflow characteristics by means of a physical model. First, a gravity current with different concentrations and discharges without using an obstacle is measured along a flume with three different slopes. Then, the height required to block the gravity current is determined by taking into account inflow characteristics and other results reflected in the literature. Finally, using the obstacle same as embankment barrier with various heights, the effects of obstacle height on the gravity current are studied in both subcritical and supercritical flows. The results specify what obstacle height is required for complete blockage of subcritical and supercritical gravity currents.

Divalent Copper Complexes as Influenza a M2 S31N Blockers

Influenza A (IAV) M2 proton channel mutations in the primary target site for amantadine cause resistance to its anti-viral drug effect. Though there is still some debate about how this mutation has caused this resistance, it is clear that novel M2 blockers are needed that are effective against the ubiquitous S31N M2 mutation. Divalent copper has previously been shown to have anti-IAV properties using in vitro assays involving block of wild type M2 channels in transfected Xenopus laevis oocytes(1) and binds to the His37 imidazoles according to solid state NMR(2). In this research, the anti-IAV property of divalent copper has been extended to organic complexes of copper. We have synthesized and purified a set of novel copper complexes of derivatized M2 blockers. We have identified a set that are stable in water, purified them of residual free copper through precipitation or crystallization as validated indirectly with inductively coupled plasma mass spec, and tested their stability in typical buffers using UV and Vis absorption spectroscopy. In two-electrode voltage clamp of Xenopus laevis oocytes transfected with A/Udorn/72 M2 S31N, some of these compounds show excellent, poorly reversible block of acid-induced inward currents with low EC50's. In addition, liposome and miniplaque assays, which were previously successful with such compounds(3), are under investigation for the refined set. 1. Gandhi et al. 1999. JBC 274:5474-5482. 2. Su et al. 2012. JACS 134:8693-8702. 3. McGuire et al, 2015. BJ 108:582a.

Quantification of the Ionic Current Contributions to Cardiac Repolarization Reserve Based on a Novel Piecewise-Linear Approximation Approach

Cardiac repolarization reserve is an important protective physiological mechanism. The assessment of single current contributions to action potential (AP) duration (APD) changes allows investigating the complex interplay of ionic mechanisms underlying such mechanism. We present a new method to quantify the contributions of each membrane current to APD changes due to a perturbation from the basal to a different condition.The method is based on a piecewise-linear approximation of the AP repolarization, which is dissected into uniform “discrete decreases” deltaV (e.g. −1 mV), splitting the repolarization timecourses into unevenly long time intervals. For each deltaV, APs are approximated by a linear decreasing trend and each current by its mean value; under this assumption the local contributions of each current to the prolongation/shortening can be computed.We tested our method on the O’Hara-Rudy (ORd) computational model of human ventricular AP in case of 50% block of the IKr current, which prolongs APD by 118 ms.Our analysis revealed that when the contribution of IKr only is considered APD results extremely prolonged (+329 ms), but other currents tend to compensate, mostly ICaL (−183 ms) and IKs to a lesser extent (−35 ms). Time-independent currents, like IK1, do not contribute to APD change. The dynamic adaptation to IKr block from the perturbation until the steady state was also quantified.Our method enables the quantification of the adaptive and compensatory mechanisms implemented by the (in silico model of) cardiac cells in response to a perturbation, such as the block of a repolarizing current.According to the ORd model, and surprisingly enough, the cardiac repolarization reserve in case of IKr block is largely attributable to a decrease in ICaL rather than to an increase in other potassium currents.