Time Constant Of Inactivation Research Articles

Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore

Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.

Characterization in Potent Modulation on Voltage-Gated Na+ Current Exerted by Deltamethrin, a Pyrethroid Insecticide

Deltamethrin (DLT) is a type-II pyrethroid ester insecticide used in agricultural and domestic applications as well as in public health. However, transmembrane ionic channels perturbed by this compound remain largely unclear, although the agent is thought to alter the gating characteristics of voltage-gated Na+ (NaV) channel current. In this study, we reappraised whether and how it and other related compounds can make any further modifications on voltage-gated Na+ current (INa) in pituitary tumor (GH3) cells. Cell exposure to DLT produced a differential and dose-dependent stimulation of peak (transient, INa(T)) or sustained (late, INa(L)) INa; consequently, the EC50 value required for DLT-stimulated INa(T) or INa(L) was determined to be 11.2 or 2.5 μM, respectively. However, neither the fast nor slow component in the inactivation time constant of INa(T) activated by short depolarizing pulse was changed with the DLT presence; conversely, tefluthrin (Tef), a type-I pyrethroid insecticide, can accentuate INa with a slowing in inactivation time course of the current. The INa(L) augmented by DLT was attenuated by further application of either dapagliflozin (Dapa) or amiloride, but not by chlorotoxin. During pulse train (PT) stimulation, with the Tef or DLT presence, the cumulative inhibition of INa(T) became slowed; moreover, following PT stimuli, a large tail current with a slowly recovering process was observed. Alternatively, during rapid depolarizing pulse, the amplitude of INa(L) and tail INa (INa(Tail)) for each depolarizing pulse became progressively increased by adding DLT, not by Tef. The recovery time constant following PT stimulation with continued presence of Tef or DLT was shortened by further addition of Dapa. The voltage-dependent hysteresis (Hys(V)) of persistent INa was differentially augmented by Tef or DLT. Taken together, the magnitude, gating, frequency dependence, as well as Hys(V) behavior of INa exerted by the presence of DLT or Tef might exert a synergistic impact on varying functional activities of excitable cells in culture or in vivo.

New 'kids' on the voltage-gated proton channel block.

So far one gene for Hv1 has been detected in studied species. The work presented by Chaves etal. in The FEBS Journal reported an 'Unexpected expansion of the voltage-gated proton channel family'. They searched for proton channel candidates and found three sequences in the genome of Aplysia californica (Ac), which were named AcHv1, AcHv2 and AcHv3. Based on electrophysiological experiments, AcHv1 and AcHv2 are voltage-gated channels. While AcHv1 behaves like Hv1 in other species, that is, it is voltage and pH-dependent, it can be inhibited by zinc and conducts protons outwardly, AcHv2 conducts protons inwards at symmetrical pH. AcHv3 constantly leaks protons, and its C-terminal part contains several cytoplasmic retention motifs. Through carefully designed and carried out electrophysiological experiments, Chaves etal. determined the biophysical parameters of all three proton channels, such as the voltage and the pH dependence, the threshold-voltage, the gating charge and the time constants of activation and inactivation. Comment on: https://doi.org/10.1111/febs.16617.

Asenapine, an atypical antipsychotic, blocks voltage-gated potassium channels in rabbit coronary artery smooth muscle cells

We investigated the effect of asenapine, a commonly used atypical antipsychotic, on voltage-dependent K+ (Kv) channels in rabbit coronary artery smooth muscle cells. Asenapine inhibited the Kv current in a concentration-dependent manner, with an half-inhibitory concentration (IC50) value of 8.59 ± 2.25 μM and Hill coefficient of 0.64 ± 0.06. Although asenapine did not affect the steady-state activation curve of Kv channels, it shifted the voltage dependence of the steady-state inactivation curve toward a more negative potential. Asenapine increased the recovery time constant of channel inactivation and produced use (state)-dependent inhibition of Kv channels at a stimulation frequency of 1 or 2 Hz. Pretreatment with the Kv1.5 subtype inhibitor DPO-1 reduced the Kv current; however, additional application of asenapine did not further inhibit the Kv current. Pretreatment with the Kv2.1 subtype inhibitor guangxitoxin and Kv7 inhibitor linopirdine also reduced the Kv current. However, additional application of asenapine further reduced the Kv current, similar to the application of asenapine alone. Asenapine induced membrane depolarization and vasoconstriction. Based on these results, we conclude that asenapine inhibits the Kv current in concentration- and use (state)-dependent manners by shifting the inactivation curve. The major target of asenapine is the Kv1.5 subtype channel.

Protective effect of low-intensity treadmill exercise against acetylcholine-calcium chloride-induced atrial fibrillation in mice.

Atrial fibrillation (AF) is the most common supraventricular arrhythmia, and it corresponds highly with exercise intensity. Here, we induced AF in mice using acetylcholine (ACh)-CaCl2 for 7 days and aimed to determine the appropriate exercise intensity (no, low, moderate, high) to protect against AF by running the mice at different intensities for 4 weeks before the AF induction by ACh-CaCl2. We examined the AF-induced atrial remodeling using electrocardiogram, patch-clamp, and immunohistochemistry. After the AF induction, heart rate, % increase of heart rate, and heart weight/body weight ratio were significantly higher in all the four AF groups than in the normal control; highest in the high-ex AF and lowest in the low-ex (lower than the no-ex AF), which indicates that low-ex treated the AF. Consistent with these changes, G protein-gated inwardly rectifying K+ currents, which were induced by ACh, increased in an exercise intensity-dependent manner and were lower in the low-ex AF than the no-ex AF. The peak level of Ca2+ current (at 0 mV) increased also in an exercise intensity-dependent manner and the inactivation time constants were shorter in all AF groups except for the low-ex AF group, in which the time constant was similar to that of the control. Finally, action potential duration was shorter in all the four AF groups than in the normal control; shortest in the high-ex AF and longest in the low-ex AF. Taken together, we conclude that low-intensity exercise protects the heart from AF, whereas high-intensity exercise might exacerbate AF.

Blockade of Voltage-Dependent K+ Channels by Benztropine, a Muscarinic Acetylcholine Receptor Inhibitor, in Coronary Arterial Smooth Muscle Cells.

We investigated the effect of the acetylcholine muscarinic receptor inhibitor benztropine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Benztropine inhibited Kv currents in a concentration-dependent manner, with an apparent IC50 value of 6.11 ± 0.80 μM and Hill coefficient of 0.62 ± 0.03. Benztropine shifted the steady-state activation curves toward a more positive potential, and the steady-state inactivation curves toward a more negative potential, suggesting that benztropine inhibited Kv channels by affecting the channel voltage sensor. Train pulse (1 or 2 Hz)-induced Kv currents were effectively reduced by the benztropine treatment. Furthermore, recovery time constants of Kv current inactivation increased significantly in response to benztropine. These results suggest that benztropine inhibited vascular Kv channels in a use (state)-dependent manner. The inhibitory effect of benztropine was canceled by pretreatment with the Kv 1.5 inhibitor, but there was no obvious change after pretreatment with Kv 2.1 or Kv7 inhibitors. In conclusion, benztropine inhibited the Kv current in a concentration- and use (state)-dependent manner. Inhibition of the Kv channels by benztropine primarily involved the Kv1.5 subtype. Restrictions are required when using benztropine to patients with vascular disease.

An efficient and scalable data analysis solution for automated electrophysiology platforms.

Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit. Historically, the effects of compounds on ion channel biophysical properties and voltage-dependent activation/inactivation could only be assessed by using low-throughput, manual patch clamp recording techniques. In recent years, automated patch clamp (APC) platforms have drastically increased in throughput. In contrast to their broad utilization in compound screening, APC platforms have rarely been used for mechanism of action studies, in large part due to the lack of sophisticated, scalable analysis methods for processing the large amount of data generated by APC platforms. In the current study, we developed a highly efficient and scalable software workflow to overcome this challenge. This method, to our knowledge the first of its kind, enables automated curve fitting and complex analysis of compound effects. Using voltage-gated sodium channels as an example, we were able to immediately assess the effects of test compounds on a spectrum of biophysical properties, including peak current, voltage-dependent steady state activation/inactivation, and time constants of activation and fast inactivation. Overall, this automated data analysis method provides a novel solution for in-depth analysis of large-scale APC data, and thus will significantly impact ion channel research and drug discovery.

Evaluation of potential anticonvulsant fluorinated N-benzamide enaminones as T-type Ca2+ channel blockers

Trifluoromethylated N-benzamide enaminones have been identified as potential anticonvulsants for the treatment of drug-resistant epilepsy. T-type Ca2+ channels are an important target for anti-seizure medications. Our laboratory has developed several fluorinated N-benzamide enaminone analogs that were evaluated by their ability to target T-type Ca2+ channels. Using whole cell voltage-clamp recordings, we identified two meta-trifluoromethyl N-benzamide enaminones with a significant inhibitory effect on T-type Ca2+ channels. These compounds had no effect on voltage-activated Na+ channels. We also evaluated the effect of the fluorinated N-benzamide enaminone analogs on the T-type Ca2+ channel subunits Cav3.2 and Cav3.3. The meta-trifluoromethyl N-benzamide enaminone lead analogs altered the steady-state inactivation of Cav3.2 T-type Ca2+ channels, which resulted in a significant increase in the inactivation recovery time of the channels. There was no effect of fluorinated N-benzamide enaminone analogs on the gating mechanism of T-type Ca2+ channels, as proven by the lack of effect on the activation and inactivation time constant of Ca2+ currents. On the contrary, the meta-trifluoromethyl N-benzamide enaminone lead analogs altered the gating mechanism of Cav3.3 T-type Ca2+ channels, as proven by the reduction in the activation and inactivation time constant of the channels. There was no effect on the inactivation kinetics of Cav3.3 T-type Ca2+ channels. The present results demonstrate that meta-substituted trifluoromethyl N-benzamide enaminone analogs target T-type Ca2+ channels by different mechanisms depending on the channel subunit. Meta-trifluoromethyl N-benzamide enaminone analogs can potentially lead to the design of more specific blockers of T-type Ca2+ channels for the treatment of epileptic seizures.

Effects of JZTX-V on the wild type Kv4.3 Expressed in HEK293T and Molecular Determinants in the Voltage-sensing Domains of Kv4.3 Interacting with JZTX-V

ABSTRACT JZTX-V is a toxin isolated from the venom of the Chinese spider Chilobrachys jingzhao. Previous studies had shown that JZTX-V could inhibit the transient outward potassium current of Kv4.2 and Kv4.3 expressed in Xenopus oocytes but had no effects on Kv1.2–1.4. However, the underlying action mechanism of JZTX-V on Kv4.3 remains unclear. In our study, JZTX-V could inhibit not only transient outward potassium currents evoked in small-sized DRG neurons but also Kv4.3-encoded currents expressed in HEK293T cells in the concentration and voltage dependence. The half maximal inhibitory concentration of JZTX-V on Kv4.3 was 9.6 ± 1.2 nM. In addition, the time course for JZTX-V inhibition and release of inhibition after washout were 15.8 ± 1.54 s and 58.8 ± 4.35 s. Electrophysiological assays indicated that 25 nM JZTX-V could shift significantly the voltage dependence of steady-state activation and steady-state inactivation to depolarization. Meanwhile, 25 nM JZTX-V decreased markedly the time constant of activation and inactivation but had no effect on the time constant of recovery from inactivation. To study the molecular determinants of Kv4.3, we performed alanine scanning on a conserved motif of Kv4.3 and assayed the affinity between mutants and JZTX-V. The results not only showed that I273, L275, V283, and F287 were molecular determinants in the conserved motif of Kv4.3 for interacting with JZTX-V but also speculated the underlying action mechanism that the hydrophobic interaction and steric effects played key roles in the binding of JZTX-V with Kv4.3. In summary, our studies have laid a scientific theoretical foundation for further research on the interaction mechanism between JZTX-V and Kv4.3.

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.

Inhibitory effect of the selective serotonin reuptake inhibitor paroxetine on human Kv1.3 channels

Paroxetine is one of the most effective selective serotonin reuptake inhibitors used to treat depressive and panic disorders that reduce the viability of human T lymphocytes, in which Kv1.3 channels are highly expressed. We examined whether paroxetine could modulate human Kv1.3 channels acutely and directly with the aim of understanding the biophysical effects and the underlying mechanisms of the drug. Kv1.3 channel proteins were expressed in Xenopus oocytes. Paroxetine rapidly inhibited the steady-state current and peak current of these channels within 6 min in a concentration-dependent manner; IC50s were 26.3 μM and 53.9 μM, respectively, and these effects were partially reversed by washout, which excluded the possibility of genomic regulation. At the same test voltage, paroxetine blockade of the steady-state currents was higher than that of the peak currents, and the inhibition of the steady-state current increased relative to the degree of depolarization. Paroxetine decreased the inactivation time constant in a concentration-dependent manner, but it did not affect the activation time constant, which resulted in the acceleration of intrinsic inactivation without changing ultrarapid activation. Blockade of Kv1.3 channels by paroxetine exhibited more rapid inhibition at higher activation frequencies showing the use-dependency of the blockade. Overall, these results show that paroxetine directly suppresses human Kv1.3 channels in an open state and accelerates the process of steady-state inactivation; thus, we have revealed a biophysical mechanism for possible acute immunosuppressive effects of paroxetine.

Refining the Identity and Role of Kv4 Channels in Mouse Substantia Nigra Dopaminergic Neurons.

Substantia nigra pars compacta (SNc) dopaminergic (DA) neurons display a peculiar electrical phenotype characterized in vitro by a spontaneous tonic regular activity (pacemaking activity), a broad action potential (AP) and a biphasic postinhibitory response. The transient A-type current (IA) is known to play a crucial role in this electrical phenotype, and so far, this current was considered to be carried exclusively by Kv4.3 potassium channels. Using Kv4.3−/− transgenic mice, we demonstrate that the constitutive loss of this channel is associated with increased exploratory behavior and impaired motor learning at the behavioral level. Consistently, it is also associated with a lack of compensatory changes in other ion currents at the cellular level. Using antigen retrieval (AR) immunohistochemistry, we then demonstrate that Kv4.2 potassium channels are also expressed in SNc DA neurons, although their contribution to IA appears significant only in a minority of neurons (∼5–10%). Using correlative analysis on recorded electrophysiological parameters and multicompartment modeling, we then demonstrate that, rather than its conductance level, IA gating kinetics (inactivation time constant) appear as the main biophysical property defining postinhibitory rebound delay and pacemaking frequency. Moreover, we show that the hyperpolarization-activated current (IH) has an opposing and complementary influence on the same firing features.

Shaker-IR K+ channel gating in heavy water: Role of structural water molecules in inactivation.

It has been reported earlier that the slow (C-type) inactivated conformation in Kv channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D2O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D2O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K+ currents in the outward and the inward directions in asymmetric solutions were not affected by a H2O/D2O exchange, negating an indirect effect of D2O on the rate of K+ rehydration. The elimination of the nonspecific effects of D2O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling.

Dexmedetomidine exhibits antiarrhythmic effects on human-induced pluripotent stem cell-derived cardiomyocytes through a Na/Ca channel-mediated mechanism.

BackgroundVentricular-like human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibit the electrophysiological characteristics of spontaneous beating. Previous studies demonstrated that dexmedetomidine (DMED), a highly selective and widely used α2-adrenoceptor agonist for sedation, analgesia, and stress management, may induce antiarrhythmic effects, especially ventricular tachycardia. However, the underlying mechanisms of the DMED-mediated antiarrhythmic effects remain to be fully elucidated.MethodsA conventional patch-clamp recording method was used to investigate the direct effects of DMED on spontaneous action potentials, pacemaker currents (If), potassium (K+) channel currents (IK1 and IKr), sodium (Na+) channel currents (INa), and calcium (Ca2+) channel currents (ICa) in ventricular-like hiPSC-CMs.ResultsDMED dose-dependently altered the frequency of ventricular-like spontaneous action potentials with a half-maximal inhibitory concentration (IC50) of 27.9 µM (n=6) and significantly prolonged the action potential duration at 90% repolarization (APD90). DMED also inhibited the amplitudes of the INa and ICa without affecting the activation and inactivation curves of these channels. DMED decreased the time constant of the Na+ and Ca2+ channel activation at potential –40 to –20 mv, and –20 mv. DMED increased the time constant of inactivation of the Na+ and Ca2+ channels. However, DMED did not affect the IK1, IKr, If, and their current-voltage relationship. The ability of DMED to decrease the spontaneous action potential frequency and the Na+ and Ca2+ channel amplitudes, were not blocked by yohimbine, idazoxan, or phentolamine.ConclusionsDMED could inhibit the frequency of spontaneous action potentials and decrease the INa and ICa of hiPSC-CMs via mechanisms that were independent of the α2-adrenoceptor, the imidazoline receptor, and the α1-adrenoceptor. These inhibitory effects on hiPSC-CMs may contribute to the antiarrhythmic effects of DMED.

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.

The effects of tegaserod, a gastrokinetic agent, on voltage-gated K+ channels in rabbit coronary arterial smooth muscle cells.

Tegaserod, a gastroprokinetic agent, is used to treat irritable bowel syndrome. Despite its extensive clinical use, little is known about the effects of tegaserod on vascular ion channels, especially K+ channels. Therefore, we examined the effects of tegaserod on voltage-gated K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch-clamp technique. Tegaserod inhibited Kv channels in a concentration-dependent manner with an IC50 value of 1.26±0.31µmol/L and Hill coefficient of 0.81±0.10. Although tegaserod had no effect on the steady-state activation curves of the Kv channels, the steady-state inactivation curve was shifted toward a more negative potential. These results suggest that tegaserod inhibits Kv channels by influencing their voltage sensors. The recovery time constant of channel inactivation was extended in the presence of tegaserod. Furthermore, application of train steps (1 and 2Hz) in the presence of tegaserod progressively increased the inhibition of Kv currents suggesting that tegaserod-induced Kv channel inhibition is use (state)-dependent. Pretreatment with a Kv1.5 subtype inhibitor suppressed the Kv current. However, additional application of tegaserod did not induce further inhibition. Pretreatment with a Kv2.1 or Kv7 inhibitor did not affect the inhibitory effect of tegaserod on Kv channels. Based on these results, we conclude that tegaserod inhibits vascular Kv channels in a concentration- and use (state)-dependent manner independent of its own functions. Furthermore, the major Kv channel target of tegaserod is the Kv1.5 subtype.

Involvement of pore helix in voltage-dependent inactivation of TRPM5 channel

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent-permeable cation channel that is activated by intracellular Ca2+. Expression of TRPM5 has been shown in taste cells, pancreas, brainstem and olfactory epithelium, and this channel is thought to be involved in controlling membrane potentials. In whole-cell patch-clamp recordings, TRPM5 exhibited voltage-dependent inactivation at negative membrane potentials and time constant of voltage-dependent inactivation of TRPM5 did not depend on the intracellular Ca2+ concentrations between 100 and 500 nM. Alanine substitution at Y913 and I916 in the pore helix of TRPM5 increased time constant of voltage-dependent inactivation. Meanwhile, voltage-dependent inactivation was reduced in TRPM5 mutants having glycine substitution at L901, Y913, Q915 and I916 in the pore helix. From these results, we conclude that the pore helix in the outer pore loop might play a role in voltage-dependent inactivation of TRPM5.

Non-dioxin-like polychlorinated biphenyl 19 has distinct effects on human Kv1.3 and Kv1.5 channels

Polychlorinated biphenyls (PCBs) are persistent and serious organic pollutants and can theoretically form 209 congeners. PCBs can be divided into two categories: dioxin-like (DL) and non-DL (NDL). NDL-PCBs, which lack aryl hydrocarbon receptor affinity, have been shown to perturb the functions of Jurkat T cells, cerebellar granule cells, and uterine cells. Kv1.3 and Kv1.5 channels are important in immune and heart functions, respectively. We investigated the acute effects of 2,2′,6-trichlorinated biphenyl (PCB19), an NDL-PCB, on the currents of human Kv1.3 and Kv1.5 channels. PCB19 acutely blocked the Kv1.3 peak currents concentration-dependently with an IC50 of ~2 μM, without changing the steady-state current. The PCB19-induced inhibition of the Kv1.3 peak current occurred rapidly and voltage-independently, and the effect was irreversible, excluding the possibility of genomic regulation. PCB19 increased the time constants of both activation and inactivation of Kv1.3 channels, resulting in the slowing down of both ultra-rapid activation and intrinsic inactivation. However, PCB19 failed to alter the steady-state curves of activation and inactivation. Regarding the Kv1.5 channel, PCB19 affected neither the peak current nor the steady-state current at the same concentrations tested in the Kv1.3 experiments, showing selective inhibition of PCB19 on the Kv1.3 than the Kv1.5. The presented data indicate that PCB19 could acutely affect the human Kv1.3 channel through a non-genomic mechanism, possibly causing toxic effects on various human physiological functions related to the Kv1.3 channel, such as immune and neural systems.

Modifications of sodium channel voltage dependence induce arrhythmia-favouring dynamics of cardiac action potentials.

Heart arrhythmia is a pathological condition where the sequence of electrical impulses in the heart deviates from the normal rhythm. It is often associated with specific channelopathies in cardiac tissue, yet how precisely the changes in ionic channels affect the electrical activity of cardiac cells is still an open question. Even though sodium channel mutations that underlie cardiac syndromes like the Long-Q-T and the Brugada-syndrome are known to affect a number of channel parameters simultaneously, previous studies have predominantly focused on the persistent late component of the sodium current as the causal explanation for an increased risk of heart arrhythmias in these cardiac syndromes. A systematic analysis of the impact of other important sodium channel parameters is currently lacking. Here, we investigate the reduced ten-Tusscher-model for single human epicardium ventricle cells and use mathematical bifurcation analysis to predict the dependence of the cardiac action potential on sodium channel activation and inactivation time-constants and voltage dependence. We show that, specifically, shifts of the voltage dependence of activation and inactivation curve can lead to drastic changes in the action potential dynamics, inducing oscillations of the membrane potential as well as bistability. Our results not only demonstrate a new way to induce multiple co-existing states of excitability (biexcitability) but also emphasize the critical role of the voltage dependence of sodium channel activation and inactivation curves for the induction of heart-arrhythmias.