Calcineurin-independent inhibition of KV1.3 by FK-506 (tacrolimus): a novel pharmacological property

2007 ◽  
Vol 292 (5) ◽  
pp. C1714-C1722 ◽  
Author(s):  
Hye Sook Ahn ◽  
Sung Eun Kim ◽  
Bok Hee Choi ◽  
Jin-Sung Choi ◽  
Myung-Jun Kim ◽  
...  
Keyword(s):  
Steady State ◽  
Chinese Hamster Ovary Cells ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Fk 506 ◽  
Inactivation Curve ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Steady State Inactivation

The interaction of FK-506 with KV1.3, stably expressed in Chinese hamster ovary cells, was investigated with the whole cell patch-clamp technique. FK-506 inhibited KV1.3 in a reversible, concentration-dependent manner with an IC50 of 5.6 μM. Rapamycin, another immunosuppressant, produced effects that were similar to those of FK-506 (IC50 = 6.7 μM). Other calcineurin inhibitors (cypermethrin or calcineurin autoinhibitory peptide) alone had no effect on the amplitude or kinetics of KV1.3. In addition, the inhibitory action of FK-506 continued, even after the inhibition of calcineurin activity. The inhibition produced by FK-506 was voltage dependent, increasing in the voltage range for channel activation. At potentials positive to 0 mV (where maximal conductance is reached), however, no voltage-dependent inhibition was found. FK-506 exhibited a strong use-dependent inhibition of KV1.3. FK-506 shifted the steady-state inactivation curves of KV1.3 in the hyperpolarizing direction in a concentration-dependent manner. The apparent dissociation constant for FK-506 to inhibit KV1.3 in the inactivated state was estimated from the concentration-dependent shift in the steady-state inactivation curve and was calculated to be 0.37 μM. Moreover, the rate of recovery from inactivation of KV1.3 was decreased. In inside-out patches, FK-506 not only reduced the current amplitude but also accelerated the rate of inactivation during depolarization. FK-506 also inhibited KV1.5 and KV4.3 in a concentration-dependent manner with IC50 of 4.6 and 53.9 μM, respectively. The present results indicate that FK-506 inhibits KV1.3 directly and that this effect is not mediated via the inhibition of the phosphatase activity of calcineurin.

scholarly journals Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism

2011 ◽  
Vol 300 (3) ◽  
pp. C567-C575 ◽  
Author(s):  
Hee Jae Kim ◽  
Hye Sook Ahn ◽  
Bok Hee Choi ◽  
Sang June Hahn
Keyword(s):  
Steady State ◽  
Inactivation Kinetics ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Voltage Range ◽  
Closed State ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Steady State Inactivation

The effects of genistein, a protein tyrosine kinase (PTK) inhibitor, on voltage-dependent K+ (Kv) 4.3 channel were examined using the whole cell patch-clamp techniques. Genistein inhibited Kv4.3 in a reversible, concentration-dependent manner with an IC50 of 124.78 μM. Other PTK inhibitors (tyrphostin 23, tyrphostin 25, lavendustin A) had no effect on genistein-induced inhibition of Kv4.3. Orthovanadate, an inhibitor of protein phosphatases, did not reverse the inhibition of Kv4.3 by genistein. We also tested the effects of two inactive structural analogs: genistin and daidzein. Whereas Kv4.3 was unaffected by genistin, daidzein inhibited Kv4.3, albeit with a lower potency. Genistein did not affect the activation and inactivation kinetics of Kv4.3. Genistein-induced inhibition of Kv4.3 was voltage dependent with a steep increase over the channel opening voltage range. In the full-activation voltage range positive to +20 mV, no voltage-dependent inhibition was found. Genistein had no significant effect on steady-state activation, but shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The Ki for the interaction between genistein and the inactivated state of Kv4.3, which was estimated from the concentration-dependent shift in the steady-state inactivation curve, was 1.17 μM. Under control conditions, closed-state inactivation was fitted to a single exponential function, and genistein accelerated closed-state inactivation. Genistein induced a weak use-dependent inhibition. These results suggest that genistein directly inhibits Kv4.3 by interacting with the closed-inactivated state of Kv4.3 channels. This effect is not mediated via inhibition of the PTK activity, because other types of PTK inhibitors could not prevent the inhibitory action of genistein.

scholarly journals Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog.

1989 ◽  
Vol 94 (5) ◽  
pp. 937-951 ◽  
Author(s):  
G Cota ◽  
E Stefani
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Voltage Dependence ◽  
Muscle Fibers ◽  
Dependent Manner ◽  
Inactivation Curve ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Intact Muscle ◽  
Ca2 Channels

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.

Effects of anandamide on potassium channels in rat ventricular myocytes: a suppression of Ito and augmentation of KATP channels

2012 ◽  
Vol 302 (6) ◽  
pp. C924-C930 ◽  
Author(s):  
Qian Li ◽  
Hui-Jie Ma ◽  
Sheng-Li Song ◽  
Min Shi ◽  
Hui-Juan Ma ◽  
...  
Keyword(s):  
Steady State ◽  
Receptor Antagonist ◽  
Potassium Current ◽  
Ventricular Myocytes ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Rat Ventricular Myocytes ◽  
Outward Potassium Current

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca2+ channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K+ channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K+ currents, including transient outward potassium current ( Ito), steady-state outward potassium current ( Iss), inward rectifier potassium current ( IK1), and ATP-sensitive potassium current ( IKATP) in isolated rat cardiac ventricular myocytes. Anandamide decreased Ito while increasing IKATP in a concentration-dependent manner but had no effect on Iss and IK1 in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of Ito to the left and shifted the recovery curve of Ito to the right. However, neither cannabinoid 1 (CB1) receptor antagonist AM251 nor CB2 receptor antagonist AM630 eliminated the inhibitory effect of anandamide on Ito. In addition, blockade of CB2 receptors, but not CB1 receptors, eliminated the augmentation effect of anandamide on IKATP. These data suggest that anandamide suppresses Ito through a non-CB1 and non-CB2 receptor-mediated pathway while augmenting IKATP through CB2 receptors in ventricular myocytes.

Bupivacaine Inhibition of L-Type Calcium Current in Ventricular Cardiomyocytes of Hamster 

1997 ◽  
Vol 87 (4) ◽  
pp. 926-934 ◽  
Author(s):  
Kenneth L. Rossner ◽  
Kenneth J. Freese
Keyword(s):  
Peak Amplitude ◽  
Ca Channel ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Time Dependencies

Background The local anesthetic bupivacaine is cardiotoxic when accidentally injected into the circulation. Such cardiotoxicity might involve an inhibition of cardiac L-type Ca2+ current (ICa,L). This study was designed to define the mechanism of bupivacaine inhibition of ICa,L. Methods Cardiomyocytes were enzymatically dispersed from hamster ventricles. Certain voltage- and time-dependencies of ICa,L were recorded using the whole-cell patch clamp method in the presence and absence of different concentrations of bupivacaine. Results Bupivacaine, in a concentration-dependent manner (10-300 microM), tonically inhibited the peak amplitude of ICa,L. The inhibition was characterized by an increase in the time of recovery from inactivation and a negative-voltage shift of the steady-state inactivation curve. The inhibition was shown to be voltage-dependent, and the peak amplitude of ICa,L could not be restored to control levels by a wash from bupivacaine. Conclusions The inhibition of ICa,L appears, in part, to result from bupivacaine predisposing L-type Ca channels to the inactivated state. Data from washout suggest that there may be two mechanisms of inhibition at work. Bupivacaine may bind with low affinity to the Ca channel and also affect an unidentified metabolic component that modulates Ca channel function.

scholarly journals Oxytocin Inhibits T-Type Calcium Current of Human Decidual Stromal Cells

2005 ◽  
Vol 90 (7) ◽  
pp. 4191-4197 ◽  
Author(s):  
Bo Liu ◽  
Stephen J. Hill ◽  
Raheela N. Khan
Keyword(s):  
Stromal Cells ◽  
Calcium Current ◽  
Elective Cesarean Section ◽  
Dependent Manner ◽  
Uterine Contractility ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Decidual Cells ◽  
Elective Cesarean

Abstract Context: Little is known about the crosstalk between the decidua and myometrium in relation to human labor. The hormone oxytocin (OT) is considered to be a key mediator of uterine contractility during parturition, exerting some of its effects through calcium channels. Objective: The objective was to characterize the effect of OT on the T-type calcium channel in human decidual stromal cells before and after the onset of labor. Design: The nystatin-perforated patch-clamp technique was used to record inward T-type calcium current (ICa(T)) from acutely dispersed decidual stromal cells obtained from women at either elective cesarean section [CS (nonlabor)] or after normal spontaneous vaginal delivery [SVD (labor)]. Setting: These studies took place at the University of Nottingham Medical School. Results: I Ca(T) of both SVD and CS cells were blocked by nickel (IC50 of 5.6 μm) and cobalt chloride (1 mm) but unaffected by nifedipine (10 μm). OT (1 nm to 3.5 μm) inhibited ICa(T) of SVD cells in a concentration-dependent manner, with a maximal inhibition of 79.0% compared with 26.2% in decidual cells of the CS group. OT-evoked reduction of ICa(T) was prevented by preincubation with the OT antagonist L371,257 in the SVD but not CS group. OT, in a concentration-dependent manner, displaced the steady-state inactivation curve for ICa(T) to the left in the SVD group with no significant effect on curves of the CS group. Conclusion: Inhibition of ICa(T) by OT in decidual cells obtained during labor may signify important functional remodeling of uterine signaling during this period.

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

2005 ◽  
Vol 289 (2) ◽  
pp. C425-C436 ◽  
Author(s):  
Bok Hee Choi ◽  
Jung-Ah Park ◽  
Kyung-Ryoul Kim ◽  
Ggot-Im Lee ◽  
Yong-Tae Lee ◽  
...  
Keyword(s):  
Actin Filament ◽  
Cytochalasin B ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Independent Manner ◽  
Concentration Dependent Manner ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk− cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC50 of 4.2 μM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC50 of 1.4 μM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 μM/s and 7.5 s−1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 μM) also inhibited an ultrarapid delayed rectifier K+ current ( IK,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous IK,ur in a cytoskeleton-independent manner as an open-channel blocker.

Coexpression with β1-subunit modifies the kinetics and fatty acid block of hH1α Na+channels

2000 ◽  
Vol 279 (1) ◽  
pp. H35-H46 ◽  
Author(s):  
Yong-Fu Xiao ◽  
Sterling N. Wright ◽  
Ging Kuo Wang ◽  
James P. Morgan ◽  
Alexander Leaf
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Monounsaturated Fatty Acids ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Α Subunit ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Steady State Inactivation

Voltage-gated cardiac Na+ channels are composed of α- and β1-subunits. In this study β1-subunit was cotransfected with the α-subunit of the human cardiac Na+ channel (hH1α) in human embryonic kidney (HEK293t) cells. The effects of this coexpression on the kinetics and fatty acid-induced suppression of Na+currents were assessed. Current density was significantly greater in HEK293t cells coexpressing α- and β1-subunits ( I Na,αβ) than in HEK293t cells expressing α-subunit alone ( I Na,α). Compared with I Na,α, the voltage-dependent inactivation and activation of I Na,αβ were significantly shifted in the depolarizing direction. In addition, coexpression with β1-subunit prolonged the duration of recovery from inactivation. Eicosapentaenoic acid [EPA, C20:5(n–3)] significantly reduced I Na,αβ in a concentration-dependent manner and at 5 μM shifted the midpoint voltage of the steady-state inactivation by −22 ± 1 mV. EPA also significantly accelerated channel transition from the resting state to the inactivated state and prolonged the recovery time from inactivation. Docosahexaenoic acid [C22:6(n–3)], α-linolenic acid [C18:3(n–3)], and conjugated linoleic acid [C18:2(n–6)] at 5 μM significantly inhibited both I Na,αβ and I Na,α.In contrast, saturated and monounsaturated fatty acids had no effects on I Na,αβ. This finding differs from the results for I Na,α, which was significantly inhibited by both saturated and unsaturated fatty acids. Our data demonstrate that functional association of β1-subunit with hH1α modifies the kinetics and fatty acid block of the Na+ channel.

scholarly journals Biophysical Characterization of Epigallocatechin-3-Gallate Effect on the Cardiac Sodium Channel Nav1.5

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 902 ◽  
Author(s):  
Mohamed-Yassine Amarouch ◽  
Han Kurt ◽  
Lucie Delemotte ◽  
Hugues Abriel
Keyword(s):  
Md Simulations ◽  
Inactivation Kinetics ◽  
Free Energy Barrier ◽  
Dependent Manner ◽  
Biophysical Characterization ◽  
Inactivation Curve ◽  
Cardiac Sodium Channel ◽  
Dependent Inhibition ◽  
Steady State Inactivation ◽  
Dose Dependent

Epigallocatechin-3-Gallate (EGCG) has been extensively studied for its protective effect against cardiovascular disorders. This effect has been attributed to its action on multiple molecular pathways and transmembrane proteins, including the cardiac Nav1.5 channels, which are inhibited in a dose-dependent manner. However, the molecular mechanism underlying this effect remains to be unveiled. To this aim, we have characterized the EGCG effect on Nav1.5 using electrophysiology and molecular dynamics (MD) simulations. EGCG superfusion induced a dose-dependent inhibition of Nav1.5 expressed in tsA201 cells, negatively shifted the steady-state inactivation curve, slowed the inactivation kinetics, and delayed the recovery from fast inactivation. However, EGCG had no effect on the voltage-dependence of activation and showed little use-dependent block on Nav1.5. Finally, MD simulations suggested that EGCG does not preferentially stay in the center of the bilayer, but that it spontaneously relocates to the membrane headgroup region. Moreover, no sign of spontaneous crossing from one leaflet to the other was observed, indicating a relatively large free energy barrier associated with EGCG transport across the membrane. These results indicate that EGCG may exert its biophysical effect via access to its binding site through the cell membrane or via a bilayer-mediated mechanism.

Resveratrol inhibits Kv2.2 currents through the estrogen receptor GPR30-mediated PKC pathway

2013 ◽  
Vol 305 (5) ◽  
pp. C547-C557 ◽  
Author(s):  
Wen-Hao Dong ◽  
Jia-Chen Chen ◽  
Yan-Lin He ◽  
Jia-Jie Xu ◽  
Yan-Ai Mei
Keyword(s):  
Molecular Mechanisms ◽  
Cerebellar Granule Cells ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Α Subunit ◽  
Inactivation Curve ◽  
Hek 293 ◽  
Nongenomic Action ◽  
Α Subunits

Resveratrol (REV) is a naturally occurring phytoalexin that inhibits neuronal K+ channels; however, the molecular mechanisms behind the effects of REV and the relevant α-subunit are not well defined. With the use of patch-clamp technique, cultured cerebellar granule cells, and HEK-293 cells transfected with the Kv2.1 and Kv2.2 α-subunits, we investigated the effect of REV on Kv2.1 and Kv2.2 α-subunits. Our data demonstrated that REV significantly suppressed Kv2.2 but not Kv2.1 currents with a fast, reversible, and mildly concentration-dependent manner and shifted the activation or inactivation curve of Kv2.2 channels. Activating or inhibiting the cAMP/PKA pathway did not abolish the inhibition of Kv2.2 current by REV. In contrast, activation of PKC with phorbol 12-myristate 13-acetate mimicked the inhibitory effect of REV on Kv2.2 by modifying the activation or inactivation properties of Kv2.2 channels and eliminated any further inhibition by REV. PKC and PKC-α inhibitor completely eliminated the REV-induced inhibition of Kv2.2. Moreover, the effect of REV on Kv2.2 was reduced by preincubation with antagonists of GPR30 receptor and shRNA for GPR30 receptor. Western blotting results indicated that the levels of PKC-α and PKC-β were significantly increased in response to REV application. Our data reveal, for the first time, that REV inhibited Kv2.2 currents through PKC-dependent pathways and a nongenomic action of the oestrogen receptor GPR30.

Differential inhibition of inflammatory cytokine release from cultured alveolar macrophages from smokers and non smokers by No2

1997 ◽  
Vol 16 (10) ◽  
pp. 577-588 ◽  
Author(s):  
Tiziana Dandrea ◽  
Ba Tu ◽  
Anders Blomberg ◽  
Thomas Sandström ◽  
Magnus Sköld ◽  
...  
Keyword(s):  
Steady State ◽  
Alveolar Macrophages ◽  
Exposure Model ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tnf Α ◽  
Dependent Inhibition ◽  
Steady State Levels ◽  
Dose Dependent

Human alveolar macrophages (AMs) obtained from smokers and non-smokers by bronchoalveolar lavage (BAL) were subjected to various concentrations of NO2 in an inverted monolayer exposure model. Culture super natants were collected 4 h after the exposure and assayed for secreted TNF-α, IL-1β, IL-8 and MIP-1α. The steady state levels of the mRNAs for these cytokines were also analysed in the cells. The adherence of BAL cells to plastic prior to exposure to the gas elevated the steady state mRNA levels of all four cytokines tested in smoker's cells and that of TNF-α and IL-1β, but not IL-8 (MIP-1α not tested), in non-smoker's cells. Interestingly, adherent cells from non-smokers released circa 15-, 3-,1.5- and 3-fold the amounts of IL-1β, IL-8, TNF-α and MIP-1α, respectively, than smoker's cells during control incubation or exposure to air. A 20 min exposure to NO2 (5 or 20 p.p.m.) did not increase the secretion of any of the cytokines from either cell type. In contrast, NO2 caused a concentration- dependent inhibition of the secretion of all cytokines except IL-1β from smoker's cells. Additionally, NO2 greatly diminished the release of all cytokines in response to further treatment with lipopolysaccharide (LPS). In contrast, only the secretion of TNF-α from non-smoker's cells was inhibited by the gas in a concentration- dependent manner, whilst LPS-induced secretion of the cytokines was not affected by the gas. The steady state levels of the respective mRNAs for each of the cytokines were not significantly affected in smoker's cells by exposure to NO2, except for a negative, dose-dependent trend in the case of TNF-α. Nitrogen dioxide also failed to elevate the levels of the mRNAs in non-smoker's cells but, again, tended to diminish the levels, particularly of IL-1β mRNA. However, exposure to the gas inhibited LPS- induced accumulation of cytokine mRNAs in smoker's cells only. The data suggest that macrophage-derived cytokine mediators of the sepsis response may not play a role in the generation of NO2-induced inflammation in the human lung. Conversely, the gas seems to non-specifically inhibit the release and/or production of cytokines, particularly from smoker's cells, at the post-transcrip tional level, and impairs the ability of the cells to increase the transcription and release of the cytokines in response to bacterial LPS. The fact that NO2 seriously impaired the already diminished capacity of smoker's cells to release several important pro-inflammatory cytokines, both under control conditions and in response to LPS, strongly suggest that the inhalation of NO2 in cigarette smoke may contribute to impairing host defence against infection in the lung.

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