Whole-cell Current Recordings Research Articles

Investigating the influence of XAV-939, a tankyrase inhibitor, on the density and gating of erg-mediated K+ currents in mouse MA-10 Leydig tumor cells

XAV-939(XAV) is a chemical compound that inhibits the activity of tankyrase. However, the precise way in which XAV alters membrane ionic currents is not well understood. In this study,our goal was to examine the impact of XAV on the ionic currents in mouse MA-10 Leydig cells, specifically focusing on the magnitude, gating properties,and voltage-dependent hysteresis of erg-mediated K+currents(IK(erg)). In our whole-cell current recordings we observed that the addition of XAV inhibited the density of IK(erg) in a concentration-dependent manner with an IC50 of 3.1 μM. Furthermore we found that continued exposure to XAV, further addition of neither liraglutide nor insulin-like growth factor-1 counteracted XAV-mediated inhibition of IK(erg). Additionally the presence of XAV suppressed the mean current versus voltage relationship of IK(erg) across the entire voltage-clamp step analyzed. This compound shifted the steady-state activation curve of IK(erg) to a less negative potential by approximately 12 mV. The presence of XAV increased the time constant of deactivating IK(erg) in MA-10 cells. The voltage-dependent clockwise hysteresis of IK(erg) responding to prolonged upright isosceles-triangular ramp voltage became diminished by adding XAV; moreover subsequent addition of NS3623 effectively reversed XAV-induced decrease of hysteretic area of IK(erg). XAV also inhibited the proliferation of this cell line and the IC50 value of XAV-induced inhibition of cell proliferation was 2.8M. Overall the suppression of IK(erg) by XAV may serve as a significant ionic mechanism that contribute to the functional properties of MA-10 cells. However, it is important to note that this effect cannot be attributed solely to the inhibition of tankyrase.

A Developmental Switch in Cholinergic Mechanisms of Modulation in the Medial Nucleus of the Trapezoid Body.

The medial nucleus of the trapezoid body (MNTB) has been intensively investigated as a primary source of inhibition in brainstem auditory circuitry. MNTB-derived inhibition plays a critical role in the computation of sound location, as temporal features of sounds are precisely conveyed through the calyx of Held/MNTB synapse. In adult gerbils, cholinergic signaling influences sound-evoked responses of MNTB neurons via nicotinic acetylcholine receptors (nAChRs; Zhang et al., 2021) establishing a modulatory role for cholinergic input to this nucleus. However, the cellular mechanisms through which acetylcholine (ACh) mediates this modulation in the MNTB remain obscure. To investigate these mechanisms, we used whole-cell current and voltage-clamp recordings to examine cholinergic physiology in MNTB neurons from Mongolian gerbils (Meriones unguiculatus) of both sexes. Membrane excitability was assessed in brain slices, in pre-hearing (postnatal days 9-13) and post-hearing onset (P18-20) MNTB neurons during bath application of agonists and antagonists of nicotinic (nAChRs) and muscarinic receptors (mAChRs). Muscarinic activation induced a potent increase in excitability most prominently prior to hearing onset with nAChR modulation emerging at later time points. Pharmacological manipulations further demonstrated that the voltage-gated K+ channel KCNQ (Kv7) is the downstream effector of mAChR activation that impacts excitability early in development. Cholinergic modulation of Kv7 reduces outward K+ conductance and depolarizes resting membrane potential. Immunolabeling revealed expression of Kv7 channels as well as mAChRs containing M1 and M3 subunits. Together, our results suggest that mAChR modulation is prominent but transient in the developing MNTB and that cholinergic modulation functions to shape auditory circuit development.

Pico145 inhibits TRPC4-mediated mICAT and postprandial small intestinal motility

In intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mICAT), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mICAT and Ca2+ signalling in mouse ileal myocytes, and on intestinal motility. Ileal myocytes freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents and for intracellular Ca2+ imaging using Fura-2. Functional assessment of Pico145’s effects was carried out by standard in vitro tensiometry, ex vivo video recordings and in vivo postprandial intestinal transit measurements using carmine red. Carbachol (50 µM)-induced mICAT was strongly inhibited by Pico145 starting from 1 pM. The IC50 value for the inhibitory effect of Pico145 on this current evoked by intracellularly applied GTPγS (200 µM), and thus lacking desensitisation, was found to be 3.1 pM, while carbachol-induced intracellular Ca2+ rises were inhibited with IC50 of 2.7 pM. In contrast, the current activated by direct TRPC4 agonist (-)-englerin A was less sensitive to the action of Pico145 that caused only ∼43 % current inhibition at 100 pM. The inhibitory effect developed rather slowly and it was potentiated by membrane depolarisation. In functional assays, Pico145 produced concentration-dependent suppression of both spontaneous and carbachol-evoked intestinal smooth muscle contractions and delayed postprandial intestinal transit. Thus, Pico145 is a potent GI-active small-molecule which completely inhibits mICAT at picomolar concentrations and which is as effective as trpc4 gene deficiency in in vivo intestinal motility tests.

De novo CLPTM1 variants with reduced GABAA R current response in patients with epilepsy.

To investigate the clinical features and potential pathogenesis mechanism of de novo CLPTM1 variants associated with epilepsy. Identify de novo genetic variants associated with epilepsy by reanalyzing trio-based whole-exome sequencing data. We analyzed the clinical characteristics of patients with these variants and performed functional in vitro studies in cells expressing mutant complementary DNAfor these variants using whole-cell voltage-clamp current recordings and outside-out patch-clamp recordings from transiently transfected human embryonic kidney (HEK) cells. Two de novo missense variants related to epilepsy were identified in the CLPTM1 gene. Functional studies indicated that CLPTM1-p.R454H and CLPTM1-p.R568Q variants reduced the γ-aminobutyric acid A receptor (GABAA R) current response amplitude recorded under voltage clamp compared to the wild-type receptors. These variants also reduced the charge transfer and altered the time course of desensitization and deactivation following rapid removal of GABA. The surface expression of the GABAA R γ2 subunit from the CLPTM1-p.R568Q group was significantly reduced compared to CLPTM1-WT. This is the first report of functionally relevant variants within the CLPTM1 gene. Patch-clamp recordings showed that these de novo CLPTM1 variants reduce GABAA R currents and charge transfer, which should promote excitation and hypersynchronous activity. This study may provide insights into the molecular mechanisms of the CLPTM1 variants underlying the patients' phenotypes, as well as for exploring potential therapeutic targets for epilepsy.

Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca2+ Channels in Rat Adrenal Medullary Chromaffin Cells.

Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types have many common properties in membrane excitability and signaling. However, AMC cells function as endocrine cells while sympathetic ganglion cells are neurons. In rat sympathetic ganglion cells, muscarinic M1 and M4 receptors mediate excitation and inhibition via suppression of M-type K+ channels and suppression of voltage-dependent Ca2+ channels, respectively. On the other hand, M1 receptor stimulation in rat AMC cells also produces excitation by suppressing TWIK-related acid sensitive K+ (TASK) channels. However, whether M4 receptors are coupled with voltage-dependent Ca2+ channel suppression is unclear. We explore this issue electrophysiologically and biochemically. Electrical stimulation of nerve fibers in rat adrenal glands trans-synaptically increased the Ca2+ signal in AMC cells. This electrically evoked increased Ca2+ signal was not altered during muscarine-induced increase in Ca2+ signal, whereas it decreased significantly during a GABA-induced increase, due to a shunt effect of increased Cl- conductance. The whole-cell current recordings revealed that voltage-dependent Ca2+ currents in AMC cells were suppressed by adenosine triphosphate, but not by muscarinic agonists. The fractionation analysis and immunocytochemistry indicated that CaV1.2 Ca2+ channels and M4 receptors are located in the raft and non-raft membrane domains, respectively. We concluded that muscarinic stimulation in rat AMC cells does not produce voltage-dependent Ca2+ channel inhibition. This lack of muscarinic inhibition is at least partly due to physical separation of voltage-dependent Ca2+ channels and M4 receptors in the plasma membrane.

Functional LTCC-β2AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure.

Beta-2 adrenergic receptors (β2ARs) but not beta-2 adrenergic receptors (β1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and β adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. Global signaling between LTCCs and β adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and β1AR or β2AR in different membrane microdomains in control and failing cardiomyocytes. LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when β2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and β2AR was lost. Interestingly, local stimulation of β1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via β1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the β2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular "global" level PKA plays a major role downstream β1AR and results in an increase in LTCC current. Regulation of the LTCC activity by proximity coupling mechanisms occurs only via β2AR, but not β1AR. This may explain how β2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.

Concerted suppressive effects of carisbamate, an anti-epileptic alkyl-carbamate drug, on voltage-gated Na+ and hyperpolarization-activated cation currents.

Carisbamate (CRS, RWJ-333369) is a new anti-seizure medication. It remains unclear whether and how CRS can perturb the magnitude and/or gating kinetics of membrane ionic currents, despite a few reports demonstrating its ability to suppress voltage-gated Na+ currents. In this study, we observed a set of whole-cell current recordings and found that CRS effectively suppressed the voltage-gated Na+ (INa) and hyperpolarization-activated cation currents (Ih) intrinsically in electrically excitable cells (GH3 cells). The effective IC50 values of CRS for the differential suppression of transient (INa(T)) and late INa (INa(L)) were 56.4 and 11.4 μM, respectively. However, CRS strongly decreased the strength (i.e., Δarea) of the nonlinear window component of INa (INa(W)), which was activated by a short ascending ramp voltage (Vramp); the subsequent addition of deltamethrin (DLT, 10 μM) counteracted the ability of CRS (100 μM, continuous exposure) to suppress INa(W). CRS strikingly decreased the decay time constant of INa(T) evoked during pulse train stimulation; however, the addition of telmisartan (10 μM) effectively attenuated the CRS (30 μM, continuous exposure)-mediated decrease in the decay time constant of the current. During continued exposure to deltamethrin (10 μM), known to be a pyrethroid insecticide, the addition of CRS resulted in differential suppression of the amplitudes of INa(T) and INa(L). The amplitude of Ih activated by a 2-s membrane hyperpolarization was diminished by CRS in a concentration-dependent manner, with an IC50 value of 38 μM. For Ih, CRS altered the steady-state I-V relationship and attenuated the strength of voltage-dependent hysteresis (Hys(V)) activated by an inverted isosceles-triangular Vramp. Moreover, the addition of oxaliplatin effectively reversed the CRS-mediated suppression of Hys(V). The predicted docking interaction between CRS and with a model of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel or between CRS and the hNaV1.7 channel reflects the ability of CRS to bind to amino acid residues in HCN or hNaV1.7 channel via hydrogen bonds and hydrophobic interactions. These findings reveal the propensity of CRS to modify INa(T) and INa(L) differentially and to effectively suppress the magnitude of Ih. INa and Ih are thus potential targets of the actions of CRS in terms of modulating cellular excitability.

Characterization of Stimulatory Action on Voltage-Gated Na+ Currents Caused by Omecamtiv Mecarbil, Known to Be a Myosin Activator.

Omecamtiv mecarbil (OM, CK-1827452) is recognized as an activator of myosin and has been demonstrated to be beneficial for the treatment of systolic heart failure. However, the mechanisms by which this compound interacts with ionic currents in electrically excitable cells remain largely unknown. The objective of this study was to investigate the effects of OM on ionic currents in GH3 pituitary cells and Neuro-2a neuroblastoma cells. In GH3 cells, whole-cell current recordings showed that the addition of OM had different potencies in stimulating the transient (INa(T)) and late components (INa(L)) of the voltage-gated Na+ current (INa) with different potencies in GH3 cells. The EC50 value required to observe the stimulatory effect of this compound on INa(T) or INa(L) in GH3 cells was found to be 15.8 and 2.3 µM, respectively. Exposure to OM did not affect the current versus voltage relationship of INa(T). However, the steady-state inactivation curve of the current was observed to shift towards a depolarized potential of approximately 11 mV, with no changes in the slope factor of the curve. The addition of OM resulted in an increase in the decaying time constant during the cumulative inhibition of INa(T) in response to pulse-train depolarizing stimuli. Furthermore, the presence of OM led to a shortening of the recovery time constant in the slow inactivation of INa(T). Adding OM also resulted in an augmentation of the strength of the window Na+ current, which was evoked by a short ascending ramp voltage. However, the OM exposure had little to no effect on the magnitude of L-type Ca2+ currents in GH3 cells. On the other hand, the delayed-rectifier K+ currents in GH3 cells were observed to be mildly suppressed in its presence. Neuro-2a cells also showed a susceptibility to the differential stimulation of INa(T) or INa(L) upon the addition of OM. Molecular analysis revealed potential interactions between the OM molecule and hNaV1.7 channels. Overall, the direct stimulation of INa(T) and INa(L) by OM is assumed to not be mediated by an interaction with myosin, and this has potential implications for its pharmacological or therapeutic actions occurring in vivo.

Non-stationary noise analysis of whole-cell currents from CHO hERG expression systems.

The modelling literature for hERG ion channels consists of many different models that do not agree well on estimates of the open probability of the channel. The current used for the estimation of the parameters has several possible sources of noise in whole-cell current recordings: stochastic gating of ion channels; biological variability in expression and regulation of subunits and signaling; and experimental artefacts. When fitting models, noise is usually simplified and modeled as i.i.d. Gaussian noise, but in this study, we look into other possible mechanistic or statistical descriptions. Having a better noise model adds information that has been neglected up to now and should lead to a more dependable model and predictions. We aim to add information from the observed noise to test and enhance models’ descriptions of open probability by improving parameter estimation using non-stationary noise theory. This theory describes stochastic gating by assuming ion channels open/close independently of each other following a binomial distribution and predicts a quadratic relationship between the mean and variance of the number of open channels. We test this theory in experimental current measurements of potassium channels (hERG) from a CHO expression line. Moreover, we also look for other possible correlations - between the variance of the mean current and the current magnitude, voltage or time. Identifying the most relevant noise sources and adding them to our likelihood function will improve the inferences about model parameters and mechanistic modelling of the ion channels.

Inhibition of Voltage-Gated Na+ Currents Exerted by KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea), an Inhibitor of Na+-Ca2+ Exchanging Process.

KB-R7943, an isothiourea derivative, has been recognized as an inhibitor in the reverse mode of the Na+-Ca2+ exchanging process. This compound was demonstrated to prevent intracellular Na+-dependent Ca2+ uptake in intact cells; however, it is much less effective at preventing extracellular Na+-dependent Ca2+ efflux. Therefore, whether or how this compound may produce any perturbations on other types of ionic currents, particularly on voltage-gated Na+ current (INa), needs to be further studied. In this study, the whole-cell current recordings demonstrated that upon abrupt depolarization in pituitary GH3 cells, the exposure to KB-R7943 concentration-dependently depressed the transient (INa(T)) or late component (INa(L)) of INa with an IC50 value of 11 or 0.9 μM, respectively. Likewise, the dissociation constant for the KB-R7943-mediated block of INa on the basis of a minimum reaction scheme was estimated to be 0.97 μM. The presence of benzamil or amiloride could suppress the INa(L) magnitude. The instantaneous window Na+ current (INa(W)) activated by abrupt ascending ramp voltage (Vramp) was suppressed by adding KB-R7943; however, subsequent addition of deltamethrin or tefluthrin (Tef) effectively reversed KB-R7943-inhibted INa(W). With prolonged duration of depolarizing pulses, the INa(L) amplitude became exponentially decreased; moreover, KB-R7943 diminished INa(L) magnitude. The resurgent Na+ current (INa(R)) evoked by a repolarizing Vramp was also suppressed by adding this compound; moreover, subsequent addition of ranolazine or Tef further diminished or reversed, respectively, its reduction in INa(R) magnitude. The persistent Na+ current (INa(P)) activated by sinusoidal voltage waveform became enhanced by Tef; however, subsequent application of KB-R7943 counteracted Tef-stimulated INa(P). The docking prediction reflected that there seem to be molecular interactions of this molecule with the hNaV1.2 or hNaV1.7 channels. Collectively, this study highlights evidence showing that KB-R7943 has the propensity to perturb the magnitude and gating kinetics of INa (e.g., INa(T), INa(L), INa(W), INa(R), and INa(P)) and that the NaV channels appear to be important targets for the in vivo actions of KB-R7943 or other relevant compounds.

The nematode serotonin-gated chloride channel MOD-1: A novel target for anthelmintic therapy

Anthelmintics are used to treat human and veterinary parasitic diseases and to reduce crop and livestock production loss associated with parasitosis. The free-living nematode Caenorhabditis elegans, a model system for anthelmintic drug discovery, has a serotonin (5-HT)-gated chloride channel, MOD-1, which belongs to the Cys-loop receptor family and modulates locomotory and behavioral functions. Since MOD-1 is unique to nematodes, it is emerging as an attractive anthelmintic drug target, but details of MOD-1 function are unclear. Here, we revealed novel aspects of MOD-1 function from the molecular level to the organism level and identified compounds targeting this receptor, which may provide new directions for anthelmintic drug discovery. We used whole-cell current recordings from heterologously expressed MOD-1 to show that tryptamine (Tryp), a weak partial agonist of vertebrate serotonin type 3 (5-HT3) receptors, efficaciously activates MOD-1. A screen for modulators revealed that GABAergic ligands piperazine (PZE) and muscimol reduce 5-HT-elicited currents, thus identifying novel MOD-1 allosteric inhibitors. Next, we performed locomotor activity assays, and we found 5-HT and Tryp rapidly decrease worm motility, which is reversible only at low 5-HT concentrations. Mutants lacking MOD-1 are partially resistant to both drugs, demonstrating its role in locomotion. Acting as an antagonist of MOD-1, we showed PZE reduces the locomotor effects of exogenous 5-HT. Therefore, Tryp- and PZE-derived compounds, acting at MOD-1 through different molecular mechanisms, emerge as promising anthelmintic agents. This study enhances our knowledge of the function and drug selectivity of Cys-loop receptors and postulates MOD-1 as a potential target for anthelmintic therapy.

Abstract P3002: Endothelial Trpm2 Deletion Attenuates Ischemic Stroke By Preventing Blood-brain Barrier Disruption

Introduction: Endothelial hyperpermeability is a hallmark of brain damage at the early stage of ischemic stroke. The subsequent blood-brain barrier (BBB) disruption drives the initial pathological changes and aggregates neuronal death. Ca 2+ overload is an important cause of endothelial dysfunction. TRPM2 is a Ca 2+ -permeable ion channel activated by oxidative stress, and oxidative stress is a critical contributor for endothelial hyperpermeability during ischemic stroke. Hypothesis: We hypothesized that TRPM2-mediated Ca 2+ influx plays a key role in causing endothelial hyperpermeability during ischemic stroke. Methods: Specific deletion of Trpm2 in endothelial cells was achieved by crossing Cdh5-cre mice with Trpm2 fl/fl mice. Middle cerebral artery occlusion (MCAO) was performed to simulate ischemic stroke. Tetrazolium chloride and Evans blue assays were used to evaluate infarct size and BBB leakage. Occludin and F4-80 / myeloperoxidase staining were used to detect tight junction degradation and immune cell invasion. Cerebral endothelial cells (CECs) were isolated and cultured. Oxygen-glucose deprivation (OGD) was used to simulate in vitro ischemic conditions. In vitro endothelial leakage assays were used to examine CEC permeability. Rhodamine-123 imaging and Ca 2+ imaging were used to evaluate oxidative stress and Ca 2+ overload in CECs. WB was used to examine CD36 signaling activation and whole-cell current recording was used to detect TRPM2 activation. Results: Specific deletion of Trpm2 in endothelial cells protects mice against MCAO-induced brain injury, which is characterized by reduced infarct size, mitigated plasma extravasation, reduced neutrophil / macrophage invasion and inhibited oxidative stress in the brain. In vitro experiments demonstrated that Trpm2 deletion alleviates ROS production, Ca 2+ overload and endothelial hyperpermeability induced by OGD and CD36 ligand thrombospondin-1 (TSP1). In CECs, activation of CD36 signaling induced by OGD and TSP1 was impaired by TRPM2 knockout. In transfected HEK293T cells, OGD and TSP1 induce the activation of TRPM2 currents in a CD36-dependent manner. Conclusion: Endothelial specific TRPM2 knockout attenuates ischemic stroke by preserving BBB integrity.

The Evidence for Sparsentan-Mediated Inhibition of INa and IK(erg): Possibly Unlinked to Its Antagonism of Angiotensin II or Endothelin Type a Receptor.

Sparsentan is viewed as a dual antagonist of endothelin type A (ETA) receptor and angiotensin II (AngII) receptor and it could be beneficial in patients with focal segmental glomerulosclerosis. Moreover, it could improve glomerular filtration rate and augment protective tissue remodeling in mouse models of focal segmental glomerulosclerosis. The ionic mechanisms through which it interacts with the magnitude and/or gating kinetics of ionic currents in excitable cells were not thoroughly investigated. Herein, we aimed to examine the effects of varying sparsentan concentrations on ionic currents residing in pituitary GH3 somatolactotrophs. From whole-cell current recordings made in GH3 cells, sparsentan (0.3–100 μM) differentially inhibited the peak and late components of voltage-gated Na+ current (INa). The IC50 value of sparsentan required to exert a reduction in peak and late INa in GH3 cells was 15.04 and 1.21 μM, respectively; meanwhile, the KD value estimated from its shortening in the slow component of INa inactivation time constant was 2.09 μM. The sparsentan (10 μM) presence did not change the overall current–voltage relationship of INa; however, the steady-state inactivation curve of the current was shifted to more negative potential in its presence (10 μM), with no change in the gating charge of the curve. The window INa activated by a brief upsloping ramp was decreased during exposure to sparsentan (10 μM); moreover, recovery of peak INa became slowed in its presence. The Tefluthrin (Tef)-stimulated resurgent INa activated in response to abrupt depolarization followed by the descending ramp pulse was additionally attenuated by subsequent application of sparsentan. In continued presence of Tef (3 μM) or β-pompilidotoxin (3 μM), further application of sparsentan (3 μM) reversed their stimulation of INa. However, sparsentan-induced inhibition of INa failed to be overcome by subsequent application of either endothelin 1 (1 μM) or angiotensin II (1 μM); moreover, in continued presence of endothelin (1 μM) or angiotensin II (1 μM), further addition of sparsentan (3 μM) effectively decreased peak INa. Additionally, the application of sparsentan (3 μM) inhibited the peak and late components of erg-mediated K+ current in GH3 cells, although it mildly decreased the amplitude of delayed-rectifier K+ current. Altogether, this study provides a distinct yet unidentified finding that sparsentan may perturb the amplitude or gating of varying ionic currents in excitable cells.

Single-Walled Carbon Nanotubes Inhibit TRPC4-Mediated Muscarinic Cation Current in Mouse Ileal Myocytes.

Single-walled carbon nanotubes (SWCNTs) are characterized by a combination of rather unique physical and chemical properties, which makes them interesting biocompatible nanostructured materials for various applications, including in the biomedical field. SWCNTs are not inert carriers of drug molecules, as they may interact with various biological macromolecules, including ion channels. To investigate the mechanisms of the inhibitory effects of SWCNTs on the muscarinic receptor cation current (mICAT), induced by intracellular GTPγs (200 μM), in isolated mouse ileal myocytes, we have used the patch-clamp method in the whole-cell configuration. Here, we use molecular docking/molecular dynamics simulations and direct patch-clamp recordings of whole-cell currents to show that SWCNTs, purified and functionalized by carboxylation in water suspension containing single SWCNTs with a diameter of 0.5–1.5 nm, can inhibit mICAT, which is mainly carried by TRPC4 cation channels in ileal smooth muscle cells, and is the main regulator of cholinergic excitation–contraction coupling in the small intestinal tract. This inhibition was voltage-independent and associated with a shortening of the mean open time of the channel. These results suggest that SWCNTs cause a direct blockage of the TRPC4 channel and may represent a novel class of TRPC4 modulators.

The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action.

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 μM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

Effective Accentuation of Voltage-Gated Sodium Current Caused by Apocynin (4'-Hydroxy-3'-methoxyacetophenone), a Known NADPH-Oxidase Inhibitor.

Apocynin (aPO, 4′-Hydroxy-3′-methoxyacetophenone) is a cell-permeable, anti-inflammatory phenolic compound that acts as an inhibitor of NADPH-dependent oxidase (NOX). However, the mechanisms through which aPO can interact directly with plasmalemmal ionic channels to perturb the amplitude or gating of ionic currents in excitable cells remain incompletely understood. Herein, we aimed to investigate any modifications of aPO on ionic currents in pituitary GH3 cells or murine HL-1 cardiomyocytes. In whole-cell current recordings, GH3-cell exposure to aPO effectively stimulated the peak and late components of voltage-gated Na+ current (INa) with different potencies. The EC50 value of aPO required for its differential increase in peak or late INa in GH3 cells was estimated to be 13.2 or 2.8 μM, respectively, whereas the KD value required for its retardation in the slow component of current inactivation was 3.4 μM. The current–voltage relation of INa was shifted slightly to more negative potential during cell exposure to aPO (10 μM); however, the steady-state inactivation curve of the current was shifted in a rightward direction in its presence. Recovery of peak INa inactivation was increased in the presence of 10 μM aPO. In continued presence of aPO, further application of rufinamide or ranolazine attenuated aPO-stimulated INa. In methylglyoxal- or superoxide dismutase-treated cells, the stimulatory effect of aPO on peak INa remained effective. By using upright isosceles-triangular ramp pulse of varying duration, the amplitude of persistent INa measured at low or high threshold was enhanced by the aPO presence, along with increased hysteretic strength appearing at low or high threshold. The addition of aPO (10 μM) mildly inhibited the amplitude of erg-mediated K+ current. Likewise, in HL-1 murine cardiomyocytes, the aPO presence increased the peak amplitude of INa as well as decreased the inactivation or deactivation rate of the current, and further addition of ranolazine or esaxerenone attenuated aPO-accentuated INa. Altogether, this study provides a distinctive yet unidentified finding that, despite its effectiveness in suppressing NOX activity, aPO may directly and concertedly perturb the amplitude, gating and voltage-dependent hysteresis of INa in electrically excitable cells. The interaction of aPO with ionic currents may, at least in part, contribute to the underlying mechanisms through which it affects neuroendocrine, endocrine or cardiac function.

Modulation of recombinant human alpha 1 glycine receptor by flavonoids and gingerols.

The inhibitory glycine receptor (GlyR) is a principal mediator of fast synaptic inhibition in mammalian spinal cord, brainstem, and higher brain centres. Flavonoids are secondary plant metabolites that exhibit many beneficial physiological effects, including modulatory action on neuronal receptors. Using whole-cell current recordings from recombinant human α1 GlyRs, expressed in HEK293cells, we compared the flavonols kaempferol and quercetin, the flavanone naringenin, the flavones apigenin and nobiletin, the isoflavone genistein, and two gingerols, 6-gingerol and 8-gingerol for their modulation of receptor currents. All compounds were inhibitors of the GlyR with IC50 values ranging between 9.3±2.6µM (kaempferol) and 46.7±6.5µM (genistein), following a mixed mode of inhibition. Co-application of two inhibitors revealed distinct binding sites for flavonoids and gingerols. Pore-lining mutants T258A and T258S were strongly inhibited by quercetin and naringenin, but not by 6-gingerol, confirming the existence of distinct binding sites for flavonoids and gingerols. Apigenin, kaempferol, nobiletin, naringenin and 6-gingerol showed biphasic action, potentiating glycine-induced currents at low concentration of both, modulator and glycine, and inhibiting at higher concentrations. Identification of distinct modulatory sites for flavonoids and related compounds may present pharmacological target sites and aid the discovery of novel glycinergic drugs.

Effectiveness of Columbianadin, a Bioactive Coumarin Derivative, in Perturbing Transient and Persistent INa.

Columbianadin (CBN) is a bioactive coumarin-type compound with various biological activities. However, the action of CBN on the ionic mechanism remains largely uncertain, albeit it was reported to inhibit voltage-gated Ca2+ current or to modulate TRP-channel activity. In this study, whole-cell patch-clamp current recordings were undertaken to explore the modifications of CBN or other related compounds on ionic currents in excitable cells (e.g., pituitary GH3 cells and HL-1 atrial cardiomyocytes). GH3-cell exposure to CBN differentially decreased peak or late component of voltage-gated Na+ current (INa) with effective IC50 of 14.7 or 2.8 µM, respectively. The inactivation time course of INa activated by short depolarization became fastened in the presence of CBN with estimated KD value of 3.15 µM. The peak INa diminished by 10 µM CBN was further suppressed by subsequent addition of either sesamin (10 µM), ranolazine (10 µM), or tetrodotoxin (1 µM), but it was reversed by 10 µM tefluthrin (Tef); however, further application of 10 µM nimodipine failed to alter CBN-mediated inhibition of INa. CBN (10 µM) shifted the midpoint of inactivation curve of INa to the leftward direction. The CBN-mediated inhibition of peak INa exhibited tonic and use-dependent characteristics. Using triangular ramp pulse, the hysteresis of persistent INa enhanced by Tef was noticed, and the behavior was attenuated by subsequent addition of CBN. The delayed-rectifier or erg-mediated K+ current was mildly inhibited by 10 µM CBN, while it also slightly inhibited the amplitude of hyperpolarization-activated cation current. In HL-1 atrial cardiomyocytes, CBN inhibited peak INa and raised the inactivation rate of the current; moreover, further application of 10 µM Tef attenuated CBN-mediated decrease in INa. Collectively, this study provides an important yet unidentified finding revealing that CBN modifies INa in electrically excitable cells.

High ability of zileuton ((±)-1-(1-benzo[b]thien-2-ylethyl)-1-hydroxyurea) to stimulate IK(Ca) but suppress IK(DR) and IK(M) independently of 5-lipoxygenase inhibition

Zileuton (Zyflo®) is regarded to be an inhibitor of 5-lipoxygenase. Although its effect on Ca2+-activated K+ currents has been reported, its overall ionic effects on neurons are uncertain. In whole-cell current recordings, zileuton increased the amplitude of Ca2+-activated K+ currents with an EC50 of 3.2 μM in pituitary GH3 lactotrophs. Furthermore, zileuton decreased the amplitudes of both delayed-rectifier K+ current (IK(DR)) and M-type K+ current (IK(M)). Conversely, no modification of hyperpolarization-activated cation current (Ih) was demonstrated in its presence of zileuton, although the subsequent addition of cilobradine effectively suppressed the current. In inside-out current recordings, the addition of zileuton to the bath increased the probability of large-conductance Ca2+-activated K+ (BKCa) channels; however, the subsequent addition of GAL-021 effectively reversed the stimulation of channel activity. The kinetic analyses showed an evident shortening in the slow component of mean closed time of BKCa channels in the presence of zileuton, with minimal change in mean open time or that in the fast component of mean closed time. The elevation of BKCa channels caused by zileuton was also observed in hippocampal mHippoE-14 neurons, without any modification of single-channel amplitude. In conclusion, except for its suppression of 5-lipoxygenase, our results indicate that zileuton does not exclusively act on BKCa channels, and its inhibitory effects on IK(DR) and IK(M) may combine to exert strong influence on the functional activities of electrically excitable cells in vivo.

Evidence for the Effectiveness of Remdesivir (GS-5734), a Nucleoside-Analog Antiviral Drug in the Inhibition of I K(M) or I K(DR) and in the Stimulation of I MEP.

Remdesivir (RDV, GS-5734), a broad-spectrum antiviral drug in the class of nucleotide analogs, has been particularly tailored for treatment of coronavirus infections. However, to which extent RDV is able to modify various types of membrane ion currents remains largely uncertain. In this study, we hence intended to explore the possible perturbations of RDV on ionic currents endogenous in pituitary GH3 cells and Jurkat T-lymphocytes. The whole-cell current recordings of ours disclosed that upon membrane depolarization in GH3 cells the exposure to RDV concentration-dependently depressed the peak or late components of I K(DR) elicitation with effective IC50 values of 10.1 or 2.8 μM, respectively; meanwhile, the value of dissociation constant of RDV-induced blockage of I K(DR) on the basis of the first-order reaction was yielded to be 3.04 μM. Upon the existence of RDV, the steady-state inactivation curve of I K(DR) was established in the RDV presence; moreover, the recovery became slowed. However, RDV-induced blockage of I K(DR) failed to be overcome by further addition of either α,β-methylene ATP or cyclopentyl-1,3-dipropylxanthine. The RDV addition also lessened the strength of M-type K+ current with the IC50 value of 2.5 μM. The magnitude of voltage hysteresis of I K(M) elicited by long-lasting triangular ramp pulse was diminished by adding RDV. Membrane electroporation-induced current in response to large hyperpolarization was enhanced, with an EC50 value of 5.8 μM. Likewise, in Jurkat T-lymphocytes, adding RDV declined I K(DR) amplitude concomitantly with the raised rate of current inactivation applied by step depolarization. Therefore, in terms of the RDV molecule, there appears to be an unintended activity of the prodrug on ion channels. Its inhibition of both I K(DR) and I K(M) occurring in a non-genomic fashion might provide additional but important mechanisms through which in vivo cellular functions are seriously perturbed.