Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue

Background and Purpose

Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied.

Experimental Approach

hERG and K_IR2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit I_Kr. Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues.

Key Results

Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and K_IR2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not K_IR2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective.

Conclusions and Implications

Drug-induced trafficking defects can be minimized if certain chemical features are avoided or ‘synthesized out’; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS.     

The 5-HT2 antagonist ketanserin is an open channel blocker of human cardiac ether-à-go-go-related gene (hERG) potassium channels

Background and purpose:

Ketanserin, a selective 5-HT receptor antagonist, prolongs the QT interval of ECG in patients. The purpose of the present study was to determine whether ketanserin would block human cardiac ether-à-go-go-related gene (hERG) potassium channels.

Experimental approach:

Whole-cell patch voltage-clamp technique was used to record membrane currents in HEK 293 cells expressing wild type or mutant hERG channel genes.

Key results:

Ketanserin blocked hERG current (I_hERG) in a concentration-dependent manner (IC₅₀=0.11 μM). The drug showed an open channel blocking property, the block increasing significantly at depolarizing voltages between +10 to +60 mV. Voltage-dependence for inactivation of hERG channels was negatively shifted by 0.3 μM ketanserin. A 2.8 fold attenuation of inhibition by elevation of external K⁺ concentration (from 5.0 to 20 mM) was observed, whereas the inactivation-deficient mutants S620T and S631A had the IC₅₀s of 0.84±0.2 and 1.7±0.4 μM (7.6 and 15.4 fold attenuation of block). In addition, the hERG mutants in pore helix and S6 also significantly reduced the channel block (2–59 fold) by ketanserin.

Conclusions and implications:

These results suggest that ketanserin binds to and blocks the open hERG channels in the pore helix and the S6 domain; channel inactivation is also involved in the blockade of hERG channels. Blockade of hERG channels most likely contributes to the prolongation of QT intervals in ECG observed clinically at therapeutic concentrations of ketanserin.     

Differential effects of short- and long-term zolpidem treatment on recombinant α1β2γ2s subtype of GABAA receptors in vitro

Aim:

Zolpidem is a non-benzodiazepine agonist at benzodiazepine binding site in GABA_A receptors, which is increasingly prescribed. Recent studies suggest that prolonged zolpidem treatment induces tolerance. The aim of this study was to explore the adaptive changes in GABA_A receptors following short and long-term exposure to zolpidem in vitro.

Methods:

Human embryonic kidney (HEK) 293 cells stably expressing recombinant α1β2γ2s GABA_A receptors were exposed to zolpidem (1 and 10 μmol/L) for short-term (2 h daily for 1, 2, or 3 consecutive days) or long-term (continuously for 48 h). Radioligand binding studies were used to determine the parameters of [³H]flunitrazepam binding sites.

Results:

A single (2 h) or repeated (2 h daily for 2 or 3 d) short-term exposure to zolpidem affected neither the maximum number of [³H]flunitrazepam binding sites nor the affinity. In both control and short-term zolpidem treated groups, addition of GABA (1 nmol/L–1 mmol/L) enhanced [³H]flunitrazepam binding in a concentration-dependent manner. The maximum enhancement of [³H]flunitrazepam binding in short-term zolpidem treated group was not significantly different from that in the control group. In contrast, long-term exposure to zolpidem resulted in significantly increase in the maximum number of [³H]flunitrazepam binding sites without changing the affinity. Furthermore, long-term exposure to zolpidem significantly decreased the ability of GABA to stimulate [³H]flunitrazepam binding.

Conclusion:

The results suggest that continuous, but not intermittent and short-term, zolpidem-exposure is able to induce adaptive changes in GABA_A receptors that could be related to the development of tolerance and dependence.     

State-dependent blockade of human ether-a-go-go- related gene （hERG） K＋ channels by changrolin in stably transfected HEK293 cells

Aim:

To study the effect of changrolin on the K⁺ channels encoded by the human ether-a-go-go-related gene (hERG).

Methods:

hERG channels were heterologously stably expressed in human embryonic kidney 293 cells, and the hERG K⁺ currents were recorded using a standard whole-cell patch-clamp technique.

Results:

Changrolin inhibited hERG channels in a concentration-dependent and reversible manner (IC₅₀=18.23 μmol/L, 95% CI: 9.27–35.9 μmol/L; Hill coefficient=−0.9446). In addition, changrolin shifted the activation curve of hERG channels by 14.3±1.5 mV to more negative potentials (P<0.01, n=9) but did not significantly affect the steady-state inactivation of hERG (n=5, P>0.05). The relative block of hERG channels by changrolin was close to zero at the time point of channel opening by the depolarizing voltage step and quickly increased afterwards. The maximal block was achieved in the inactivated state, with no further development of the open channel block. In the “envelope of tails” experiments, the time constants of activation were found to be 287.8±46.2 ms and 174.2±18.4 ms, respectively, for the absence and presence of 30 μmol/L changrolin (P<0.05, n=7). The onset of inactivation was accelerated significantly by changrolin between −40 mV and +60 mV (P<0.05, n=7).

Conclusion:

The results demonstrate that changrolin is a potent hERG blocker that preferentially binds to hERG channels in the open and inactivated states.     

High potency inhibition of hERG potassium channels by the sodium-calcium exchange inhibitor KB-R7943

BACKGROUND AND PURPOSE

KB-R7943 is an isothiourea derivative that is used widely as a pharmacological inhibitor of sodium–calcium exchange (NCX) in experiments on cardiac and other tissue types. This study investigated KB-R7943 inhibition of hERG (human ether-à-go-go-related gene) K⁺ channels that underpin the cardiac rapid delayed rectifier potassium current, I_Kr.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp measurements were made of hERG current (I_hERG) carried by wild-type or mutant hERG channels and of native rabbit ventricular I_Kr. Docking simulations utilized a hERG homology model built on a MthK-based template.

KEY RESULTS

KB-R7943 inhibited both I_hERG and native I_Kr rapidly on membrane depolarization with IC₅₀ values of ∼89 and ∼120 nM, respectively, for current tails at −40 mV following depolarizing voltage commands to +20 mV. Marked I_hERG inhibition also occurred under ventricular action potential voltage clamp. I_hERG inhibition by KB-R7943 exhibited both time- and voltage-dependence but showed no preference for inactivated over activated channels. Results of alanine mutagenesis and docking simulations indicate that KB-R7943 can bind to a pocket formed of the side chains of aromatic residues Y652 and F656, with the compound''s nitrobenzyl group orientated towards the cytoplasmic side of the channel pore. The structurally related NCX inhibitor SN-6 also inhibited I_hERG, but with a markedly reduced potency.

CONCLUSIONS AND IMPLICATIONS

KB-R7943 inhibits I_hERG/I_Kr with a potency that exceeds that reported previously for acute cardiac NCX inhibition. Our results also support the feasibility of benzyloxyphenyl-containing NCX inhibitors with reduced potential, in comparison with KB-R7943, to inhibit hERG.     

Epidermal growth factor receptor tyrosine kinase regulates the human inward rectifier potassium K(IR)2.3 channel, stably expressed in HEK 293 cells

BACKGROUND AND PURPOSE

The detailed molecular modulation of inward rectifier potassium channels (including the K_IR2.3 channel) is not fully understood. The present study was designed to determine whether human K_IR2.3 (K_IR2.3) channels were regulated by protein tyrosine kinases (PTKs).

EXPERIMENTAL APPROACH

Whole-cell patch voltage-clamp, immunoprecipitation, Western blot analysis and site-directed mutagenesis were employed to determine the potential PTK phosphorylation of Kir2.3 current in HEK 293 cells stably expressing Kir2.3 gene.

KEY RESULTS

The broad-spectrum PTK inhibitor genistein (10 µM) and the selective epidermal growth factor (EGF) kinase inhibitor AG556 (10 µM) reversibly decreased K_IR2.3 current and the effect was reversed by the protein tyrosine phosphatase inhibitor, orthovanadate (1 mM). Although EGF (100 ng·mL⁻¹) and orthovanadate enhanced K_IR2.3 current, this effect was antagonized by AG556. However, the Src-family tyrosine kinase inhibitor PP2 (10 µM) did not inhibit K_IR2.3 current. Tyrosine phosphorylation of K_IR2.3 channels was decreased by genistein or AG556, and was increased by EGF or orthovanadate. The decrease of tyrosine phosphorylation of K_IR2.3 channels by genistein or AG556 was reversed by orthovanadate or EGF. Interestingly, the response of K_IR2.3 channels to EGF or AG556 was lost in the K_IR2.3 Y234A mutant channel.

CONCLUSION AND IMPLICATIONS

These results demonstrate that the EGF receptor tyrosine kinase up-regulates the K_IR2.3 channel via phosphorylation of the Y234 residue of the WT protein. This effect may be involved in the endogenous regulation of cellular electrical activity.     

Does terfenadine-induced ventricular tachycardia/fibrillation directly relate to its QT prolongation and Torsades de Pointes?

BACKGROUND AND PURPOSE

Terfenadine has been reported to cause cardiac death. Hence, we investigated its pro-arrhythmic potential in various in vitro models.

EXPERIMENTAL APPROACH

Pro-arrhythmic effects of terfenadine were investigated in rabbit isolated hearts and left ventricular wedge preparations. Also, using whole-cell patch-clamp recording, we examined its effect on the human ether-à-go-go-related gene (hERG) current in HEK293 cells transfected with hERG and on the I_Na current in rabbit ventricular cells and human atrial myocytes.

KEY RESULTS

Terfenadine concentration- and use-dependently inhibited I_Na in rabbit myocytes and in human atrial myocytes and also inhibited the hERG. In both the rabbit left ventricular wedge and heart preparations, terfenadine at 1 µM only slightly prolonged the QT- and JT-intervals but at 10 µM, it caused a marked widening of the QRS complex, cardiac wavelength shortening, incidences of in-excitability and non-TdP-like ventricular tachycardia/fibrillation (VT/VF) without prolongation of the QT/JT-interval. At 10 µM terfenadine elicited a lower incidence of early afterdepolarizations versus non- Torsades de Pointes (TdP)-like VT/VF (100% incidence), and did not induce TdPs. Although the concentration of terfenadine in the tissue-bath was low, it accumulated within the heart tissue.

CONCLUSION AND IMPLICATIONS

Our data suggest that: (i) the induction of non-TdP-like VT/VF, which is caused by slowing of conduction via blockade of I_Na (like Class Ic flecainide), may constitute a more important risk for terfenadine-induced cardiac death; (ii) although terfenadine is a potent hERG blocker, the risk for non-TdP-like VT/VF exceeds the risk for TdPs; and (iii) cardiac wavelength (λ) could serve as a biomarker to predict terfenadine-induced VT/VF.     

Robust anti-arrhythmic efficacy of verapamil and flunarizine against dofetilide-induced TdP arrhythmias is based upon a shared and a different mode of action

BACKGROUND AND PURPOSE

The high predisposition to Torsade de Pointes (TdP) in dogs with chronic AV-block (CAVB) is well documented. The anti-arrhythmic efficacy and mode of action of Ca²⁺ channel antagonists, flunarizine and verapamil against TdP were investigated.

EXPERIMENTAL APPROACH

Mongrel dogs with CAVB were selected based on the inducibility of TdP with dofetilide. The effects of flunarizine and verapamil were assessed after TdP and in different experiments to prevent dofetilide-induced TdP. Electrocardiogram and ventricular monophasic action potentials were recorded. Electrophysiological parameters and short-term variability of repolarization (STV) were determined. In vitro, flunarizine and verapamil were added to determine their effect on (i) dofetilide-induced early after depolarizations (EADs) in canine ventricular myocytes (VM); (ii) diastolic Ca²⁺ sparks in RyR2^R4496+/+ mouse myocytes; and (iii) peak and late I_Na in SCN5A-HEK 293 cells.

KEY RESULTS

Dofetilide increased STV prior to TdP and in VM prior to EADs. Both flunarizine and verapamil completely suppressed TdP and reversed STV to baseline values. Complete prevention of TdP was achieved with both drugs, accompanied by the prevention of an increase in STV. Suppression of EADs was confirmed after flunarizine. Only flunarizine blocked late I_Na. Ca²⁺ sparks were reduced with verapamil.

CONCLUSIONS AND IMPLICATIONS

Robust anti-arrhythmic efficacy was seen with both Ca²⁺ channel antagonists. Their divergent electrophysiological actions may be related to different additional effects of the two drugs.     

Modulation of mouse gastrointestinal motility by allyl isothiocyanate, a constituent of cruciferous vegetables (Brassicaceae): evidence for TRPA1-independent effects

BACKGROUND AND PURPOSE

Allyl isothiocyanate (AITC, mustard oil), a constituent of many common cruciferous vegetables (Brassicaceae), activates transient receptor potential of ankyrin type-1 (TRPA1) channels, claimed to regulate gastrointestinal contractility. In this study, we have investigated the effect of AITC on intestinal motility.

EXPERIMENTAL APPROACH

Effects of AITC were investigated in vivo on upper gastrointestinal transit in mice and in mouse isolated ileum [contractions induced by electrical field stimulation (EFS), acetylcholine and spontaneous contractility]. The contractor activity of AITC was studied in mouse isolated colon. The ability of TRPA1 channel antagonists to block AITC-induced elevation of intracellular Ca²⁺[Ca²⁺]_i was assessed in HEK293 cells transfected with rat TRPA1 channels.

KEY RESULTS

AITC increased [Ca²⁺]_i in HEK293 cells, reduced ileal contractility (acetylcholine-, EFS-induced contractions and spontaneous contractility), but contracted the isolated colon. Gentamicin and camphor (non-selective TRPA1 channel antagonists), HC-030031 and AP18 (selective TRPA1 channel agonists) inhibited AITC-induced effects in HEK293 cells but not in the ileum or colon. AITC-induced contractions were reduced by tetrodotoxin and strongly reduced by nifedipine, cyclopiazonic acid and ryanodine. In vivo, AITC reduced (following i.p. administration) or increased (following intragastric administration) upper gastrointestinal transit in mice These effects were not affected by HC-030031.

CONCLUSION AND IMPLICATIONS

AITC, depending, in vitro, on the regions of gut examined and, in vivo, on the route of administration, exerted both stimulatory and inhibitory effects on intestinal motility, which were not sensitive to TRPA1 channel antagonists. The proposition that TRPA1 channels are the primary targets for AITC to induce contraction should be revised.     

Pharmacology of the short QT syndrome N588K-hERG K+ channel mutation: differential impact on selected class I and class III antiarrhythmic drugs

Background and purpose:

The short QT syndrome (SQTS) is associated with cardiac arrhythmias and sudden death. The SQT1 form of SQTS results from an inactivation-attenuated, gain-of-function mutation (N588K) to the human ether-à-go-go-related gene (hERG) potassium channel. Pharmacological blockade of this mutated hERG channel may have therapeutic value. However, hERG-blocking potencies of canonical inhibitors such as E-4031 and D-sotalol are significantly reduced for N588K-hERG. Here, five hERG-blocking drugs were compared to determine their relative potencies for inhibiting N588K channels, and two other inactivation-attenuated mutant channels were tested to investigate the association between impaired inactivation and altered drug potency.

Experimental approach:

Whole-cell patch clamp measurements of hERG current (I_hERG) mediated by wild-type and mutant (N588K, S631A and N588K/S631A) channels were made at 37 °C CHO cells.

Key results:

The N588K mutation attenuated I_hERG inhibition in the following order: E-4031>amiodarone>quinidine>propafenone>disopyramide. Comparing the three inactivation mutants, the two single mutations, although occurring in different modules of the channel, attenuated inactivation to a nearly identical degree, whereas the double mutant caused considerably greater attenuation, permitting the titration of inactivation. Attenuation of channel inhibition was similar between the single mutants for each drug, and was significantly greater with the double mutant.

Conclusions and implications:

The degree of drug inhibition of hERG channels may vary based on the level of channel inactivation. Drugs previously identified as useful for treating SQT1 have the least dependence on hERG inactivation. In addition, our findings indicate that amiodarone may warrant further investigation as a potential treatment for SQT1.     

Smooth muscle relaxation and activation of the large conductance Ca++ – activated K+ (BKCa) channel by novel oestrogens

Background and Purpose

Oestrogens can interact directly with membrane receptors and channels and can activate vascular BK_Ca channels. We hypothesized that novel oestrogen derivatives could relax smooth muscle by an extracllular effect on the α and β₁ subunits of the BK_Ca channel, rather than at an intracellular site.

Experimental Approach

We studied the effects of novel oestrogens on the tension of pre-contracted isolated rat aortic rings, and on the electrophysiological properties of HEK 293 cells expressing the hSloα or hSloα+β₁ subunits. Two of the derivatives incorporated a quaternary ammonium side-chain making them membrane impermeable.

Key Results

Oestrone, oestrone oxime and Quat DME-oestradiol relaxed pre-contracted rat aorta, but only Quat DME-oestradiol-induced relaxation was iberiotoxin sensitive. However, only potassium currents recorded in HEK 293 cells over-expressing both hSloα and hSloβ₁ were activated by oestrone, oestrone oxime and Quat DME-oestradiol.

Conclusion and Implications

The novel oestrogens were able to relax smooth muscle, but through different mechanisms. In particular, oestrone oxime required the presence of the endothelium to exert much of its effect, whilst Quat DME-oestradiol depended both on NO and BK_Ca channel activation. The activation of BK_Ca currents in HEK 293 cells expressing hSloα+β₁ by Quat DME-oestradiol is consistent with an extracellular binding site between the two subunits. The binding site resides between the extracellular N terminal of the α subunit and the extracellular loop between TM1 and 2 of the β₁ subunit. Membrane-impermeant Quat DME-oestradiol lacks an exchangeable hydrogen on the A ring obviating antioxidant activity.     

Activation of transient receptor potential A1 by a non-pungent capsaicin-like compound, capsiate

BACKGROUND AND PURPOSE

Capsiate is produced by ‘CH-19 Sweet’ (Capsicum annuun L.), a non-pungent cultivar of red pepper. Like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system and suppressing inflammation, but the underlying mechanisms for this are uncertain. We previously reported that capsiate could activate transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor. The purpose of the present study is to investigate whether capsinoids activate other TRP channels.

EXPERIMENTAL APPROACH

Using Ca²⁺ imaging and whole-cell patch-clamp methods, we analysed the response of TRP channels to three kinds of capsinoids, capsiate, dihydrocapsiate and nordihydrocapsiate, in HEK293T cells expressing TRP channels or in primary cultures of mouse dorsal root ganglion neurons.

KEY RESULTS

We found that in both cell types TRP ankyrin 1 (TRPA1) had a slightly weaker response to capsinoids compared with TRPV1, with the capsiate EC₅₀ for TRPA1 activation being more than that for TRPV1 activation, and that the capsinoid-evoked action was blocked by a specific TRPA1 antagonist. TRPA1 was activated by capsinoids, but not by their degradation products. Amino acids known to participate in TRPA1 activation following cysteine covalent modification or zinc treatment were not involved in the activation of TRPA1 by capsinoid.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that capsinoids activate TRPA1 by an as yet unknown mechanism, and TRPA1 could be involved in physiological phenomena associated with capsinoid treatment.     

Effect of K+ and Rb+ on the action of verapamil on a voltage-gated K+ channel,hKv1.3: implications for a second open state?

Background and purpose:

Verapamil blocks current through the voltage-gated K⁺ channel K_v1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity.

Experimental“ approach:

We investigated the influence of intra- and extracellular K⁺ and Rb⁺ on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hK_v1.3 channels.

Key results:

Verapamil affinity was highest in high intracellular K⁺ concentrations ([K⁺]_i) and lowest in low [Rb⁺]_i, indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K⁺ and Rb⁺ could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate.

Conclusions and implications:

Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hK_v1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state.     

The protease inhibitor atazanavir blocks hERG K+ channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane

Aim:

Atazanavir (ATV) is a HIV-1 protease inhibitor for the treatment of AIDS patients, which is recently reported to provoke excessive prolongation of the QT interval and torsades de pointes (TdP). In order to elucidate its arrhythmogenic mechanisms, we investigated the effects of ATV on the hERG K⁺ channels expressed in HEK293 cells.

Methods:

hERG K⁺ currents were detected using whole-cell patch clamp recording in HEK293 cells transfected with EGFP-hERG plasmids. The expression of hERG protein was measured with Western blotting. Two mutants (Y652A and F656C) were constructed in the S6 domain within the inner helices of hERG K⁺ channels that were responsible for binding of various drugs. The trafficking of hERG protein was studied with confocal microscopy.

Results:

Application of ATV (0.01–30 μmol/L) concentration-dependently decreased hERG K⁺ currents with an IC₅₀ of 5.7±1.8 μmol/L. ATV (10 μmol/L) did not affect the activation and steady-state inactivation of hERG K⁺ currents. Compared with the wild type hERG K⁺ channels, both Y652A and F656C mutants significantly reduced the inhibition of ATV on hERG K⁺ currents. Overnight treatment with ATV (0.1–30 μmol/L) concentration-dependently reduced the amount of fully glycosylated 155 kDa hERG protein without significantly affecting the core-glycosylated 135 kDa hERG protein in the cells expressing the WT-hERG protein. Confocal microscopy studies confirmed that overnight treatment with ATV obstructed the trafficking of hERG protein to the cell membrane.

Conclusion:

ATV directly blocks hERG K⁺ channels via binding to the residues Y652 and F656 in the S6 domain, and indirectly obstructs the transport of the hERG protein to the cell membrane.     

Control of TMEM16A by INO-4995 and other inositolphosphates

Background And Purpose

Ca²⁺-dependent Cl⁻ secretion (CaCC) in airways and other tissues is due to activation of the Cl⁻ channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca²⁺-activated Cl⁻ channels are regulated by membrane lipid inositol phosphates, and that 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester (INO-4995) augments CaCC. Here we examined whether TMEM16A is the target for INO-4995 and if the channel is regulated by inositol phosphates.

Experimental Approach

The effects of INO-4995 on CaCC were examined in overexpressing HEK293, colonic and primary airway epithelial cells as well as Xenopus oocytes. We used patch clamping, double electrode voltage clamp and Ussing chamber techniques.

Key Results

We found that INO-4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9 nS pF^–1 in overexpressing cells. The tetrakisphosphates Ins(3,4,5,6)P₄ or Ins(1,3,4,5)P₄ and enzymes controlling levels of InsP₄ or PIP₂ and PIP₃ had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in Xenopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, Cl⁻ currents were not acutely activated by INO-4995. However incubation with INO-4995 augmented 1.6- to 4-fold TMEM16A-dependent Cl⁻ currents activated by ionomycin or ATP, while intracellular Ca²⁺ signals were not affected. The potentiating effect of INO-4995 on transient ATP-activated TMEM16A-currents in cystic fibrosis (CF) airways was twice of that observed in non-CF airways.

Conclusions And Implications

These data indicate that TMEM16A is the target for INO-4995, although the mode of action appears different for overexpressed and endogenous channels. INO-4995 may be useful for the treatment of CF lung disease.     

Tanshinone II-A sodium sulfonate (DS-201) enhances human BKCa channel activity by selectively targeting the pore-forming α subunit

Aim:

Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BK_Ca channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BK_Ca channels.

Methods:

Human BK_Ca channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BK_Ca currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BK_Ca subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy.

Results:

DS-201 (40–160 μmol/L) concentration-dependently increased the total open probability of BK_Ca channels in HEK293 cells, associated with enhancements of Ca²⁺ and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC₅₀ for BK_Ca channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity.

Conclusion:

DS-201 selectively targets the pore-forming α subunit of human BK_Ca channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.     

Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

BACKGROUND AND PURPOSE

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.

EXPERIMENTAL APPROACH

We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.

KEY RESULTS

TRPM7 currents were inhibited by modulators of small conductance Ca²⁺-activated K⁺ channels (K_Ca2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K_Ca2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.     

The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels

BACKGROUND

Depletion of the Ca²⁺ store by ryanodine receptor (RyR) agonists induces store-operated Ca²⁺ entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE.

EXPERIMENTAL APPROACH

SOCE and ORAI1-3 currents were recorded by Ca²⁺ imaging and whole-cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1-3.

KEY RESULTS

4-CEP induced a significant release of Ca²⁺ in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration-dependent and more potent than its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca²⁺ release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly. Moreover, 4-CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca²⁺ store-depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co-expressing STIM1/ORAI1-3.

CONCLUSION AND IMPLICATIONS

4-CEP not only acts as a RyR agonist to deplete the Ca²⁺ store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1-3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists.     

Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue

BACKGROUND AND PURPOSE

Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I_f or I_h). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels.

EXPERIMENTAL APPROACH

HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings.

KEY RESULTS

In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I_f by 67% at −120 mV, while MEL57A (3 µM) reduced I_f by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I_h by 60% at −80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization.

CONCLUSIONS

Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.     

Allocryptopine and benzyltetrahydropalmatine block hERG potassium channels expressed in HEK293 cells

Aim:

Allocryptopine (ALL) is an alkaloid extracted from Corydalis decumbens (Thunb) Pers. Papaveraceae, whereas benzyltetrahydropalmatine (BTHP) is a derivative of tetrahydropalmatine extracted from Corydalis ambigua (Pall) Cham et Schlecht. The aim of this study was to investigate the effects of ALL and BTHP on the human ether-a-go-go related gene (hERG) current expressed in HEK293 cells.

Methods:

Cultured HEK293 cells were transiently transfected with hERG channel cDNA plasmid pcDNA3.1 using Lipofectamine. The whole-cell current IHERG was evoked and recorded using Axon MultiClamp 700B amplifier. The drugs were applied via supserfusion.

Results:

Both ALL and BTHP reversibly suppressed the amplitude and density of I_HERG in concentration- and voltage-dependent manners (the respective IC₅₀ value was 49.65 and 22.38 μmol/L). BTHP (30 μmol/L) caused a significant negative shift of the steady-state inactivation curve of I_HERG, while ALL (30 μmol/L) did not affect the steady-state inactivation of I_HERG. Furthermore, BTHP, but not ALL, shortened the time constants of fast inactivation and slow time constants of deactivation of I_HERG. But both the drugs markedly lengthened the time constants for recovery of I_HERG from inactivation. Using action potential waveform pulses, it was found that both the drugs at 30 μmol/L significantly suppressed the current densities in the late phase of action potential, but did not significantly affect the current densities in the early phase of action potential.

Conclusion:

Both ALL and BTHP derived from Chinese herbs potently block hERG current.