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

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00258.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. C1714-C1722 ◽

Cited By ~ 11

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.

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

Molecules ◽

10.3390/molecules25040902 ◽

2020 ◽

Vol 25 (4) ◽

pp. 902 ◽

Cited By ~ 1

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.

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

The Journal of General Physiology ◽

10.1085/jgp.94.5.937 ◽

1989 ◽

Vol 94 (5) ◽

pp. 937-951 ◽

Cited By ~ 14

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.

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

Anesthesiology ◽

10.1097/00000542-199710000-00028 ◽

1997 ◽

Vol 87 (4) ◽

pp. 926-934 ◽

Cited By ~ 26

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.

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Biophysical characterization of Epigallocatechin-3-Gallate effect on the cardiac sodium channel Nav1.5

10.31730/osf.io/zt439 ◽

2019 ◽

Author(s):

Mohamed Yassine AMAROUCH ◽

Mehmet Hanifi 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

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

AJP Cell Physiology ◽

10.1152/ajpcell.00450.2004 ◽

2005 ◽

Vol 289 (2) ◽

pp. C425-C436 ◽

Cited By ~ 5

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.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.1.h35 ◽

2000 ◽

Vol 279 (1) ◽

pp. H35-H46 ◽

Cited By ~ 44

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.

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

Human & Experimental Toxicology ◽

10.1177/096032719701601005 ◽

1997 ◽

Vol 16 (10) ◽

pp. 577-588 ◽

Cited By ~ 14

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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RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-019-02338-4 ◽

2019 ◽

Vol 472 (1) ◽

pp. 3-25 ◽

Cited By ~ 3

Author(s):

Nadine J. Ortner ◽

Alexandra Pinggera ◽

Nadja T. Hofer ◽

Anita Siller ◽

Niels Brandt ◽

...

Keyword(s):

Hair Cells ◽

Splice Variant ◽

Glutamate Release ◽

Inactivation Kinetics ◽

Dependent Manner ◽

Voltage Range ◽

Inner Hair Cells ◽

Whole Cell Patch Clamp ◽

Dependent Inactivation ◽

Voltage Dependent

AbstractCav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel’s activation threshold toward more hyperpolarized potentials. Taken together, the association with “slow” β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

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Propofol Inhibits Cardiac L-Type Calcium Current in Guinea Pig Ventricular Myocytes

Anesthesiology ◽

10.1097/00000542-199603000-00018 ◽

1996 ◽

Vol 84 (3) ◽

pp. 626-635 ◽

Cited By ~ 50

Author(s):

Ching-Yue Yang ◽

Chih-Shung Wong ◽

Chuan-Cheng Yu ◽

Hsiang-Ning Luk ◽

Cheng-I Lin

Keyword(s):

Steady State ◽

Guinea Pig ◽

Time Course ◽

Single Channel ◽

Ventricular Myocytes ◽

Hill Coefficient ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Inactivation Curve ◽

Whole Cell

Background Propofol may exert negative inotropic and chronotropic actions in the heart. Single-channel studies show that propofol affects the kinetics of opening and closing of cardiac L-type calcium channels (ICa(L)) without altering channel conductance. The aim of this study was to investigate the mechanisms of depressant effects of propofol on cardiac whole-cell ICa(L). Methods Single ventricular myocytes were freshly dissciated from guinea pig hearts using enzymatic isolation. One-suction electrode voltage-clamp technique (whole-cell mode) was used. LCa(L) was separated from other contaminated ionic currents. Propofol was applied in the commercial 10% Intralipid emulsion formula (Zeneca, UK). Results In isolated cardiomyocytes, propofol significantly inhibited whole-cell ICa(L) in a concentration-dependent manner (K D = 52.0 microM; Hill coefficient = 1.3). The solvent (Intralipid) did not affect ICa(L). Propofol decreased ICa(L) at all potentials tested along the voltage axis and reduced the slope conductance. The threshold potential for activation and the peak potential of the current-voltage relationship were not changed by propofol. The steady-state activation curves overlapped in the absence and the presence of 56 microM propofol. In contrast, the steady-state inactivation curve was shifted in the hyperpolarizing direction. The time course of the recovery from inactivation was delayed by 56 microM propofol. The blocking action on ICa(L) of propofol shows marked resting block and use-dependent block. Propofol caused more pronounced inhibition at a higher stimulation frequency. The effect of propofol on the inactivation process was even more clear on ICa(L). Conclusions The authors conclude tha propofol, at supratherapeutic concentrations, inhibits cardiac ICa(L). This inhibition is mainly due to a shift of inactivation curve and a reduction in slope conductance.

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