Use-dependent Manner Research Articles

Electron Microscopic Mapping of Mitochondrial Morphology in the Cochlear Nerve Fibers.

To enable nervous system function, neurons are powered in a use-dependent manner by mitochondria undergoing morphological-functional adaptation. In a well-studied model system-the mammalian cochlea, auditory nerve fibers (ANFs) display distinct electrophysiological properties, which is essential for collectively sampling acoustic information of a large dynamic range. How exactly the associated mitochondrial networks are deployed in functionally differentiated ANFs remains scarcely interrogated. Here, we leverage volume electron microscopy and machine-learning-assisted image analysis to phenotype mitochondrial morphology and distribution along ANFs of full-length in the mouse cochlea inner spiral bundle. This reveals greater variance in mitochondrial size with increased ANF habenula to terminal path length. Particularly, we analyzed the ANF terminal-residing mitochondria, which are critical for local calcium uptake during sustained afferent activities. Our results suggest that terminal-specific enrichment of mitochondria, in addition to terminal size and overall mitochondrial abundance of the ANF, correlates with heterogenous mitochondrial contents of the terminal.

Male Stressed Mice Having Behavioral Control Exhibit Escalations in Dorsal Dentate Adult-Born Neurons and Spatial Memory.

An escapable (ES)/inescapable stress (IS) paradigm was used to study whether behavioral control and repeated footshock stressors may affect adult neurogenesis and related cognitive function. Male stressed mice having behavioral control (ES) had a short-term escalation in dorsal dentate gyrus (DG) neurogenesis, while similarly stressed mice having no such control had unaltered neurogenesis as compared to control mice receiving no stressors. Paradoxically, ES and IS mice had comparable stress-induced corticosterone elevations throughout the stress regimen. Appetitive operant conditioning and forced running procedures were used to model learning and exercise effects in this escapable/inescapable paradigm. Further, conditioning and running procedures did not seem to affect the mice's corticosterone or short-term neurogenesis. ES and IS mice did not show noticeable long-term changes in their dorsal DG neurogenesis, gliogenesis, local neuronal density, apoptosis, autophagic flux, or heterotypic stress responses. ES mice were found to have a greater number of previously labeled and functionally integrated DG neurons as compared to IS and control mice 6 weeks after the conclusion of the stressor regimen. Likewise, ES mice outperformed IS and non-stressed control mice for the first two, but not the remaining two, trials in the object location task. Compared to non-stressed controls, temozolomide-treated ES and IS mice having a lower number of dorsal DG 6-week-old neurons display poor performance in their object location working memory. These results, taken together, prompt us to conclude that repeated stressors, albeit their corticosterone secretion-stimulating effect, do not necessary affect adult dorsal DG neurogenesis. Moreover, stressed animals having behavioral control may display adult neurogenesis escalation in the dorsal DG. Furthermore, the number of 6-week-old and functionally-integrated neurons in the dorsal DG seems to confer the quality of spatial location working memory. Finally, these 6-week-old, adult-born neurons seem to contribute spatial location memory in a use-dependent manner.

Polysorbate 80 blocked a peripheral sodium channel, Nav1.7, and reduced neuronal excitability.

Polysorbate 80 is a non-ionic detergent derived from polyethoxylated sorbitan and oleic acid. It is widely used in pharmaceuticals, foods, and cosmetics as an emulsifier. Nav1.7 is a peripheral sodium channel that is highly expressed in sympathetic and sensory neurons, and it plays a critical role in determining the threshold of action potentials (APs). We found that 10 μg/mL polysorbate 80 either abolished APs or increased the threshold of the APs of dorsal root ganglions. We thus investigated whether polysorbate 80 inhibits Nav1.7 sodium current using a whole-cell patch-clamp recording technique. Polysorbate 80 decreased the Nav1.7 current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 250.4 μg/mL at a holding potential of -120 mV. However, the IC50 was 1.1 μg/mL at a holding potential of -90 mV and was estimated to be 0.9 μg/mL at the resting potentials of neurons, where most channels are inactivated. The activation rate and the voltage dependency of activation of Nav1.7 were not changed by polysorbate 80. However, polysorbate 80 caused hyperpolarizing shifts in the voltage dependency of the steady-state fast inactivation curve. The blocking of Nav1.7 currents by polysorbate 80 was not reversible at a holding potential of -90 mV but was completely reversible at -120 mV, where the channels were mostly in the closed state. Polysorbate 80 also slowed recovery from inactivation and induced robust use-dependent inhibition, indicating that it is likely to bind to and stabilize the inactivated state. Our results indicate that polysorbate 80 inhibits Nav1.7 current in concentration-, state-, and use-dependent manners when used even below commercial concentrations. This suggests that polysorbate 80 may be helpful in pain medicine as an excipient. In addition, in vitro experiments using polysorbate 80 with neurons should be conducted with caution.

Block of Voltage-Gated Sodium Channels by Aripiprazole in a State-Dependent Manner.

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5-1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.

Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2.

The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.

Movement-dependent electrical stimulation for volitional strengthening of cortical connections in behaving monkeys

Correlated activity of neurons can lead to long-term strengthening or weakening of the connections between them. In addition, the behavioral context, imparted by execution of physical movements or the presence of a reward, can modulate the plasticity induced by Hebbian mechanisms. In the present study, we have combined behavior and induced neuronal correlations to strengthen connections in the motor cortex of adult behaving monkeys. Correlated activity was induced using an electrical-conditioning protocol in which stimuli gated by voluntary movements were used to produce coactivation of neurons at motor-cortical sites involved in those movements. Delivery of movement-dependent stimulation resulted in small increases in the strength of associated cortical connections immediately after conditioning. Remarkably, when paired with further repetition of the movements that gated the conditioning stimuli, there were substantially larger gains in the strength of cortical connections, which occurred in a use-dependent manner, without delivery of additional conditioning stimulation. In the absence of such movements, little change was observed in the strength of motor-cortical connections. Performance of the motor behavior in the absence of conditioning also did not produce any changes in connectivity. Our results show that combining movement-gated stimulation with further natural use of the "conditioned" pathways after stimulation ends can produce use-dependent strengthening of connections in adult primates, highlighting an important role for behavior in cortical plasticity. Our data also provide strong support for combining movement-gated stimulation with use-dependent physical rehabilitation for strengthening connections weakened by a stroke or spinal cord injury.

Influence of land use on the microbiological properties of urban soils

Advancing towards sustainable cities needs a better understanding of all components of urban ecosystems, including soils, but the biological component of urban soils remains poorly studied compared to natural and agricultural soils. With the objective of advancing knowledge in this aspect, we studied the microbiological properties in 61 soils of the city of Santiago de Compostela (NW Spain), under several land uses (urban grassland, urban forest, urban garden, and periurban arable land). We determined thirteen extracellular enzymatic activities as a proxy of microbial community functioning, along with measures of microbial activity (basal respiration), microbial abundance (DNA extraction and real-time PCR), and the composition of microbial communities (Illumina MiSeq sequencing on 16S rRNA and ITS marker regions). Results showed a high variability in all of the microbiological variables among soils, reflecting the typical spatial heterogeneity of urban soils. Respiration and enzymatic activities were highly correlated to soil organic matter contents but not affected by land use within the city, with the exception of alkaline phosphomonoesterase activity, which was higher in urban garden soils and correlated positively to pH and availability of phosphorus and nitrogen. Unlike fungal abundance, we recorded a higher bacterial abundance in the urban grasslands than in the other land uses. While the composition of bacterial communities was structured in a more homogeneous, land use-dependent manner (33% variance, pAdonis = 0.001), the respective fungal communities were more heterogeneous and less influenced by group-dependent characteristics (18%, pAdonis = 0.001). Soil pH had a larger influence on the bacterial community composition (28% variance) compared to the fungal composition (8.5% variance). Overall, these findings provide evidence that the typically high soil heterogeneity of urban ecosystems is the main driving force for the urban soil microbiome, with soil organic matter determining largely microbial activity.

The Effects of Structural Alterations in the Polyamine and Amino Acid Moieties of Philanthotoxins on Nicotinic Acetylcholine Receptor Inhibition in the Locust, Schistocerca gregaria.

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust (Schistocerca gregaria) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC50 = 0.80 μM at −75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC50 (half-maximal inhibitory concentration) = 0.13 μM at −75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC50 = 0.44 μM at −75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC50 = 1.71 nM at −75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 μM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

Block of Voltage-Gated Sodium Channels as a Potential Novel Anti-cancer Mechanism of TIC10.

Background: Tumor therapeutics are aimed to affect tumor cells selectively while sparing healthy ones. For this purpose, a huge variety of different drugs are in use. Recently, also blockers of voltage-gated sodium channels (VGSCs) have been recognized to possess potentially beneficial effects in tumor therapy. As these channels are a frequent target of numerous drugs, we hypothesized that currently used tumor therapeutics might have the potential to block VGSCs in addition to their classical anti-cancer activity. In the present work, we have analyzed the imipridone TIC10, which belongs to a novel class of anti-cancer compounds, for its potency to interact with VGSCs. Methods: Electrophysiological experiments were performed by means of the patch-clamp technique using heterologously expressed human heart muscle sodium channels (hNav1.5), which are among the most common subtypes of VGSCs occurring in tumor cells. Results: TIC10 angular inhibited the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state was weak with an extrapolated Kr of about 600 μM. TIC10 most probably did not interact with fast inactivation. In protocols for slow inactivation, a half-maximal inhibition occurred around 2 µM. This observation was confirmed by kinetic studies indicating that the interaction occurred with a slow time constant. Furthermore, TIC10 also interacted with the open channel with an affinity of approximately 4 µM. The binding site for local anesthetics or a closely related site is suggested as a possible target as the affinity for the well-characterized F1760K mutant was reduced more than 20-fold compared to wild type. Among the analyzed derivatives, ONC212 was similarly effective as TIC10 angular, while TIC10 linear more selectively interacted with the different states. Conclusion: The inhibition of VGSCs at low micromolar concentrations might add to the anti-tumor properties of TIC10.

Cannabidiol Inhibition of Murine Primary Nociceptors: Tight Binding to Slow Inactivated States of Nav1.8 Channels.

The nonpsychoactive phytocannabinoid cannabidiol (CBD) has been shown to have analgesic effects in animal studies but little is known about its mechanism of action. We examined the effects of CBD on intrinsic excitability of primary pain-sensing neurons. Studying acutely dissociated capsaicin-sensitive mouse DRG neurons at 37°C, we found that CBD effectively inhibited repetitive action potential firing, from 15-20 action potentials evoked by 1 s current injections in control to 1-3 action potentials with 2 μm CBD. Reduction of repetitive firing was accompanied by a reduction of action potential height, widening of action potentials, reduction of the afterhyperpolarization, and increased propensity to enter depolarization block. Voltage-clamp experiments showed that CBD inhibited both TTX-sensitive and TTX-resistant (TTX-R) sodium currents in a use-dependent manner. CBD showed strong state-dependent inhibition of TTX-R channels, with fast binding to inactivated channels during depolarizations and slow unbinding on repolarization. CBD alteration of channel availability at various voltages suggested that CBD binds especially tightly [Kd (dissociation constant), ∼150 nm] to the slow inactivated state of TTX-R channels, which can be substantially occupied at voltages as negative as -40 mV. Remarkably, CBD was more potent in inhibiting TTX-R channels and inhibiting action potential firing than the local anesthetic bupivacaine. We conclude that CBD might produce some of its analgesic effects by direct effects on neuronal excitability, with tight binding to the slow inactivated state of Nav1.8 channels contributing to effective inhibition of repetitive firing by modest depolarizations.SIGNIFICANCE STATEMENT Cannabidiol (CBD) has been shown to inhibit pain in various rodent models, but the mechanism of this effect is unknown. We describe the ability of CBD to inhibit repetitive action potential firing in primary nociceptive neurons from mouse dorsal root ganglia and analyze the effects on voltage-dependent sodium channels. We find that CBD interacts with TTX-resistant sodium channels in a state-dependent manner suggesting particularly tight binding to slow inactivated states of Nav1.8 channels, which dominate the overall inactivation of Nav1.8 channels for small maintained depolarizations from the resting potential. The results suggest that CBD can exert analgesic effects in part by directly inhibiting repetitive firing of primary nociceptors and suggest a strategy of identifying compounds that bind selectively to slow inactivated states of Nav1.8 channels for developing effective analgesics.

Block of Voltage-Gated Sodium Channels by Atomoxetine in a State- and Use-dependent Manner.

Atomoxetine, a neuroactive drug, is approved for the treatment of attention-deficit/hyperactivity disorder (ADHD). It is primarily known as a high affinity blocker of the noradrenaline transporter, whereby its application leads to an increased level of the corresponding neurotransmitter in different brain regions. However, the concentrations used to obtain clinical effects are much higher than those which are required to block the transporter system. Thus, off-target effects are likely to occur. In this way, we previously identified atomoxetine as blocker of NMDA receptors. As many psychotropic drugs give rise to sudden death of cardiac origin, we now tested the hypothesis whether atomoxetine also interacts with voltage-gated sodium channels of heart muscle type in clinically relevant concentrations. Electrophysiological experiments were performed by means of the patch-clamp technique at human heart muscle sodium channels (hNav1.5) heterogeneously expressed in human embryonic kidney cells. Atomoxetine inhibited sodium channels in a state- and use-dependent manner. Atomoxetine had only a weak affinity for the resting state of the hNav1.5 (Kr: ∼ 120 µM). The efficacy of atomoxetine strongly increased with membrane depolarization, indicating that the inactivated state is an important target. A hallmark of this drug was its slow interaction. By use of different experimental settings, we concluded that the interaction occurs with the slow inactivated state as well as by slow kinetics with the fast-inactivated state. Half-maximal effective concentrations (2–3 µM) were well within the concentration range found in plasma of treated patients. Atomoxetine also interacted with the open channel. However, the interaction was not fast enough to accelerate the time constant of fast inactivation. Nevertheless, when using the inactivation-deficient hNav1.5_I408W_L409C_A410W mutant, we found that the persistent late current was blocked half maximal at about 3 µM atomoxetine. The interaction most probably occurred via the local anesthetic binding site. Atomoxetine inhibited sodium channels at a similar concentration as it is used for the treatment of ADHD. Due to its slow interaction and by inhibiting the late current, it potentially exerts antiarrhythmic properties.

Characterization of Vixotrigine, a Broad-Spectrum Voltage-Gated Sodium Channel Blocker.

Voltage-gated sodium channels (Navs) are promising targets for analgesic and antiepileptic therapies. Although specificity between Nav subtypes may be desirable to target specific neural types, such as nociceptors in pain, many broadly acting Nav inhibitors are clinically beneficial in neuropathic pain and epilepsy. Here, we present the first systematic characterization of vixotrigine, a Nav blocker. Using recombinant systems, we find that vixotrigine potency is enhanced in a voltage- and use-dependent manner, consistent with a state-dependent block of Navs. Furthermore, we find that vixotrigine potently inhibits sodium currents produced by both peripheral and central nervous system Nav subtypes, with use-dependent IC50 values between 1.76 and 5.12 μM. Compared with carbamazepine, vixotrigine shows higher potency and more profound state-dependent inhibition but a similar broad spectrum of action distinct from Nav1.7- and Nav1.8-specific blockers. We find that vixotrigine rapidly inhibits Navs and prolongs recovery from the fast-inactivated state. In native rodent dorsal root ganglion sodium channels, we find that vixotrigine shifts steady-state inactivation curves. Based on these results, we conclude that vixotrigine is a broad-spectrum, state-dependent Nav blocker. SIGNIFICANCE STATEMENT: Vixotrigine blocks both peripheral and central voltage-gated sodium channel subtypes. Neurophysiological approaches in recombinant systems and sensory neurons suggest this block is state-dependent.

Norquetiapine blocks the human cardiac sodium channel Nav1.5 in a state-dependent manner

Quetiapine, an atypical antipsychotic drug, is used for the treatment of schizophrenia and acute mania. Although a previous report showed that quetiapine blocked hERG potassium current, quetiapine has been considered relatively safe in terms of cardiovascular side effects. In the present study, we used the whole-cell patch-clamp technique to investigate the effect that quetiapine and its major metabolite norquetiapine can exert on human cardiac sodium channels (hNav1.5). The half-maximal inhibitory concentrations of quetiapine and norquetiapine at a holding potential of −90 mV near the resting potential of cardiomyocytes were 30 and 6 μM, respectively. Norquetiapine as well as quetiapine was preferentially bound in the inactivated state of the hNav1.5 channel. Norquetiapine slowed the recovery from inactivation of hNav1.5 and consequently induced strong use-dependent inhibition. Our results indicate that norquetiapine blocks hNav1.5 current in concentration-, state- and use-dependent manners, suggesting that the blockade of hNav1.5 current by norquetiapine may shorten the cardiac action potential duration and reduce the risk of QT interval prolongation induced by the inhibition of hERG potassium currents.

Etomidate reduces excitability of the neurons and suppresses the function of nAChR ventral horn in the spinal cord of neonatal rats

To investigate the effects of etomidate on electrophysiological properties and nicotinic acetylcholine receptors (nAChRs) of ventral horn neurons in the spinal cord. The spinal cord containing lumbosacral enlargement was isolated from 19 neonatal SD rats aged 7-12 days. The spinal cord were sliced and digested with papain (0.18 g/30 mL artificial cerebrospinal fluid) and incubated for 40 min. At the ventral horn, acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes, and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons. In current-clamp mode, the spontaneous action potential (AP) of the ventral horn neurons in the spinal cord was recorded. The effects of pretreatment with different concentrations of etomidate on AP recorded in the ventral horn neurons were examined. In the voltage-clamp mode, nicotine was applied to induce inward currents in the ventral horn neurons, and the effect of pretreatment with etomidate on the inward currents induced by nicotine were examined with different etomidate concentrations, different holding potentials and different use time. The isolated ventral horn neurons were in good condition with large diverse somata and intact processes. The isolated spinal ventral horn neurons (n=21) had spontaneous action potentials, and were continuously perfused for 2 min with 0.3, 3.0 and 30.0 μmol/L etomidate. Compared with those before administration, the AP amplitude, spike potential amplitude and overshoot were concentration-dependently suppressed (P < 0.01), and spontaneous discharge frequency was obviously reduced (P < 0.01, n=12). The APs of the other 9 neurons were completely abolished by etomidate at 3.0 or 30 μmol/L. At the same holding potential (VH=-70 mV), pretreatment with 0.3, 3.0 or 30.0 μmol/L etomidate for 2 min concentration-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P < 0.01, n=7). At the holding potentials of - 30, - 50, and - 70 mV, pretreatment with 30.0 μmol/L etomidate for 2 min voltage-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P < 0.01, n=6 for each holding potential). During the 6 min of 30.0 μmol/L etomidate pretreatment, the clamped cells were exposed to 0.4 mmol/L nicotine for 4 times at 0, 2, 4, and 6 min (each exposure time was 2 s), and the nicotinic current amplitude decreased gradually as the number of exposures increased. But at the same concentration, two nicotine exposures (one at the beginning and the other at the end of the 6 min pretreatment) resulted in a significantly lower inhibition rate compared with 4 nicotine exposures (P < 0.01, n=6). etomidate reduces the excitability of the spinal ventral neurons in a concentration-dependent manner and suppresses the function of nAChR in a concentration-, voltage-, and use-dependent manner.

Inhibition of voltage-gated Na+ currents by eleclazine in rat atrial and ventricular myocytes

BackgroundAtrial-ventricular differences in voltage-gated Na+ currents might be exploited for atrial-selective antiarrhythmic drug action for the suppression of atrial fibrillation without risk of ventricular tachyarrhythmia. Eleclazine (GS-6615) is a putative antiarrhythmic drug with properties similar to the prototypical atrial-selective Na+ channel blocker ranolazine that has been shown to be safe and well tolerated in patients.ObjectiveThe present study investigated atrial-ventricular differences in the biophysical properties and inhibition by eleclazine of voltage-gated Na+ currents.MethodsThe fast and late components of whole-cell voltage-gated Na+ currents (respectively, INa and INaL) were recorded at room temperature (∼22°C) from rat isolated atrial and ventricular myocytes.ResultsAtrial INa activated at command potentials ∼5.5 mV more negative and inactivated at conditioning potentials ∼7 mV more negative than ventricular INa. There was no difference between atrial and ventricular myocytes in the eleclazine inhibition of INaL activated by 3 nM ATX-II (IC50s ∼200 nM). Eleclazine (10 μM) inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated state block. Eleclazine produced voltage-dependent instantaneous inhibition in atrial and ventricular myocytes; it caused a negative shift in voltage of half-maximal inactivation and slowed the recovery of INa from inactivation in both cell types.ConclusionsDifferences exist between rat atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage dependence of INa activation/inactivation in atrial myocytes underlies differences between the 2 cell types in the voltage dependence of instantaneous inhibition by eleclazine. Eleclazine warrants further investigation as an atrial-selective antiarrhythmic drug.

Spontaneous and evoked neurotransmission are partially segregated at inhibitory synapses

Synaptic transmission is initiated via spontaneous or action-potential evoked fusion of synaptic vesicles. At excitatory synapses, glutamatergic receptors activated by spontaneous and evoked neurotransmission are segregated. Although inhibitory synapses also transmit signals spontaneously or in response to action potentials, they differ from excitatory synapses in both structure and function. Therefore, we hypothesized that inhibitory synapses may have different organizing principles. We report picrotoxin, a GABAAR antagonist, blocks neurotransmission in a use-dependent manner at rat hippocampal synapses and therefore can be used to interrogate synaptic properties. Using this tool, we uncovered partial segregation of inhibitory spontaneous and evoked neurotransmission. We found up to 40% of the evoked response is mediated through GABAARs which are only activated by evoked neurotransmission. These data indicate GABAergic spontaneous and evoked neurotransmission processes are partially non-overlapping, suggesting they may serve divergent roles in neuronal signaling.

Protriptyline, a tricyclic antidepressant, inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

In this study, we explore the inhibitory effects of protriptyline, a tricyclic antidepressant drug, on voltage-dependent K+ (Kv) channels of rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Protriptyline inhibited the vascular Kv current in a concentration-dependent manner, with an IC50 value of 5.05 ± 0.97μM and a Hill coefficient of 0.73 ± 0.04. Protriptyline did not affect the steady-state activation kinetics. However, the drug shifted the steady-state inactivation curve to the left, suggesting that protriptyline inhibited the Kv channels by changing their voltage sensitivity. Application of 20 repetitive train pulses (1 or 2Hz) progressively increased the protriptyline-induced inhibition of the Kv current, suggesting that protriptyline inhibited Kv channels in a use (state)-dependent manner. The extent of Kv current inhibition by protriptyline was similar during the first, second, and third step pulses. These results suggest that protriptyline-induced inhibition of the Kv current mainly occurs principally in the closed state. The increase in the inactivation recovery time constant in the presence of protriptyline also supported use (state)-dependent inhibition of Kv channels by the drug. In the presence of the Kv1.5 inhibitor, protriptyline did not induce further inhibition of the Kv channels. However, pretreatment with a Kv2.1 or Kv7 inhibitor induced further inhibition of Kv current to a similar extent to that observed with protriptyline alone. Thus, we conclude that protriptyline inhibits the vascular Kv channels in a concentration- and use-dependent manner by changing their gating properties. Furthermore, protriptyline-induced inhibition of Kv channels mainly involves the Kv1.5.

Eleclazine Inhibition of Voltage-gated Sodium Currents in Rat Atrial and Ventricular Myocytes

Atrial-ventricular differences in voltage-gated Na+ currents might be exploited for atrial-selective antiarrhythmic drug action for the suppression of atrial fibrillation without risk of ventricular tachyarrhythmia side-effects. Eleclazine (GS-6615) is a putative antiarrhythmic drug that has been shown to be safe and well-tolerated in patients and has properties similar to the prototypical atrial-selective Na+ channel blocker, ranolazine.Atrial-ventricular differences in the biophysical properties of voltage-gated Na+ currents and their inhibition by eleclazine were investigated.The fast and late components of whole-cell voltage-gated Na+ currents (respectively, INa & INaL) were recorded at room temperature (∼22 °C) from rat isolated atrial and ventricular myocytes. Atrial INa activated at command potentials ∼5.5 mV more negative and inactivated at conditioning potentials ∼7 mV more negative than ventricular INa. Under control conditions, INaL density was ∼2-fold greater in atrial than in ventricular myocytes. There was no difference between the two cell types in block of INaL by eleclazine (IC50s ∼200 nM) in the presence of the INaL activator, ATX-II (3 nM). 10 μM eleclazine inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated state block. Eleclazine produced an instantaneous inhibition of INa in atrial but not ventricular myocytes; the drug caused a negative shift in voltage of half-maximal inactivation and slowed the recovery of INa from inactivation in both cell types. Thus, differences exist between rat atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage-dependence of INa activation/inactivation underlies the instantaneous block by eleclazine in atrial myocytes. Eleclazine warrants further investigation as an atrial-selective antiarrhythmic drug.

Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10−12 to 10−5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

Inhibitory effect of immunosuppressive drug tacrolimus on voltage-gated K+ current in rabbit coronary arterial smooth muscle cells

In the present study, we investigated the inhibitory effect of tacrolimus, a macrolide immunosuppressive drug that acts through calcineurin inhibition, on voltage-gated K+ (Kv) channels expressed in native smooth muscle cells isolated from the coronary arteries of rabbits. Tacrolimus reduced the amplitude of Kv currents in a dose-dependent manner with an IC50 value and Hill coefficient of 7.80 ± 3.01 μM and 1.07 ± 0.25, respectively. Tacrolimus caused a shift in the activation curve toward a more positive potential and in the inactivation curve toward a more negative potential. Tacrolimus-induced inhibition of Kv current was increased by the application of train pulses (1 or 2 Hz). Furthermore, the recovery time constant of inactivation was extended in the presence of tacrolimus, suggesting that tacrolimus inhibited Kv channels in a use-dependent manner. Two kinds of Kv subtype inhibitors, DPO-1 and guangxitoxin did not affect the degree of tacrolimus-induced inhibition of Kv current. Furthermore, pretreatment with another calcineurin inhibitor, cyclosporine A, did not affect the Kv current, and did not alter the inhibitory effect of tacrolimus. Using perforated-patch clamp experiments, inhibition of Kv channels by tacrolimus caused membrane depolarization. From these results, we concluded that tacrolimus inhibited the vascular Kv currents in a dose, state (open and closed)-dependent manner.