Y3+ Block Demonstrates an Intracellular Activation Gate for the α1G T-type Ca2+ Channel

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular “pore gate” has also been proposed. We have used the voltage dependence of block by extracellular Y3+ as a tool to locate the activation gate of the α1G (CaV3.1) T-type calcium channel. Y3+ block exhibited no clear voltage dependence from −40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y3+ at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca2+ was replaced with Ba2+, the rate of open block by Y3+ was unaffected, but closed block was threefold faster than in Ca2+, suggesting the slower closed-block rate reflects ion–ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter

Ni2+ Block of CaV3.1 (α1G) T-type Calcium Channels

Ni2+ inhibits current through calcium channels, in part by blocking the pore, but Ni2+ may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni2+ on CaV3.1 is affected by permeant ions. We find two components to block by Ni2+, a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent Kd = 3 vs. 1 mM Ni2+, with 2 mM Ca2+ or Ba2+) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca2+ or Ba2+). Slow block depends both on the concentration and on the identity of the permeant ion (Ca2+ vs. Ba2+ vs. Na+). Slow block is 2–3× faster in Ba2+ than in Ca2+ (2 or 110 mM), and is ∼10× faster with 2 vs. 110 mM Ca2+ or Ba2+. Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations (∼3 μM Ca2+). We conclude that both fast and slow block of CaV3.1 by Ni2+ are most consistent with occlusion of the pore. The exit rate of Ni2+ for slow block is reduced at high Ni2+ concentrations, suggesting that the site responsible for fast block can “lock in” slow block by Ni2+, at a site located deeper within the pore. In contrast to the complex pore block observed for CaV3.1, inhibition of CaV3.2 by Ni2+ was essentially independent of voltage, and was similar in 2 mM Ca2+ vs. Ba2+, consistent with inhibition by a different mechanism, at a site outside the pore

Permeation and Gating in CaV3.1 (α1G) T-type Calcium Channels Effects of Ca2+, Ba2+, Mg2+, and Na+

We examined the concentration dependence of currents through CaV3.1 T-type calcium channels, varying Ca2+ and Ba2+ over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current–voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by PCa/PNa = 87 and PCa/PBa = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent Kd values were similar for Ca2+ and Ba2+, both for block of currents carried by Na+ (3 μM for Ca2+ vs. 4 μM for Ba2+, at −30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca2+ vs. 2.5 mM for Ba2+; nearly voltage independent). Block by 3–10 μM Ca2+ was time dependent, described by bimolecular kinetics with binding at ∼3 × 108 M−1s−1 and voltage-dependent exit. Ca2+o, Ba2+o, and Mg2+o also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e− per 98 Å2 from Gouy-Chapman theory. Additionally, inward currents inactivated ∼35% faster in Ba2+o (vs. Ca2+o or Na+o). The accelerated inactivation in Ba2+o correlated with the transition from Na+ to Ba2+ permeation, suggesting that Ba2+o speeds inactivation by occupying the pore. We conclude that the selectivity of the “surface charge” among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca2+ or Ba2+

Nonclinical cardiovascular safety of pitolisant: comparing International Conference on Harmonization S7B and Comprehensive in vitro Pro-arrhythmia Assay initiative studies

Background and purpose: We evaluated the concordance of results from two sets of nonclinical cardiovascular safety studies on pitolisant. Experimental approach: Nonclinical studies envisaged both in the ICH S7B guideline and Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were undertaken. CiPA-initiative studies included in vitro ion channels and stem cell-derived human ventricular myocyte studies as well as in silico modelling of results to simulate human ventricular electrophysiology. ICH S7B-recommended studies included in vitro hERG studies, in vivo dog study with follow-up investigations in rabbit Purkinje fibres and in vivo studies in the Carlsson rabbit proarrhythmia model. Key results: Both sets of nonclinical studies consistently excluded pitolisant from having clinically relevant QT-liability or proarrhythmic potential. CiPA studies revealed pitolisant to have modest calcium channel blocking and late I Na reducing activities at high concentrations, which resulted in reduction of dofetilide-induced early after-depolarisations (EADs) by pitolisantin ICH S7B studies. Studies in stem cell-derived human cardiomyocytes with dofetilide or E-4031 given alone and in combination with pitolisant confirmed these properties. In silico modelling confirmed that the measured ion channel effects are consistent with results from both the stem cell-derived cardiomyocyte and rabbit Purkinje fibre studies and categorised pitolisant as a drug with low torsadogenic potential. The results from the two sets of nonclinical studies correlated well with two clinical QT studies. Conclusions and implications: Our experience supports the CiPA initiative but suggests that sponsors should consider investigating drug effects on EADs and the use of proarrhythmia models when the results from CiPA studies are ambiguous

Epigallocatechin-3-gallate induces mesothelioma cell death via H2O2-dependent T-type Ca2+ channel opening

Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin-3-gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H2O2release in cell cultures, and exogenous catalase (CAT) abrogated EGCG-induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3-loaded, EGCG-exposed MMe cells showed significant [Ca2+]irise, prevented by CAT, dithiothreitol or the T-type Ca2+channel blockers mibefradil and NiCl2. Cell loading with dihydrorhodamine 123 revealed EGCG-induced ROS production, prevented by CAT, mibefradil or the Ca2+chelator BAPTA-AM. Direct exposure of cells to H2O2produced similar effects on Ca2+and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Cav3.2 T-type Ca2+channels in these cells, compared to normal mesothelium. Also, Cav3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Cav3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T-type Ca2+channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target. \ua9 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd

Tailoring mathematical models to stem-cell derived cardiomyocyte lines can improve predictions of drug-induced changes to their electrophysiology

Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have applications in disease modeling, cell therapy, drug screening and personalized medicine. Computational models can be used to interpret experimental findings in iPSC-CMs, provide mechanistic insights, and translate these findings to adult cardiomyocyte (CM) electrophysiology. However, different cell lines display different expression of ion channels, pumps and receptors, and show differences in electrophysiology. In this exploratory study, we use a mathematical model based on iPSC-CMs from Cellular Dynamic International (CDI, iCell), and compare its predictions to novel experimental recordings made with the Axiogenesis Cor.4U line. We show that tailoring this model to the specific cell line, even using limited data and a relatively simple approach, leads to improved predictions of baseline behavior and response to drugs. This demonstrates the need and the feasibility to tailor models to individual cell lines, although a more refined approach will be needed to characterize individual currents, address differences in ion current kinetics, and further improve these results

A malignant hyperthermia–inducing mutation in RYR1 (R163C): alterations in Ca2+ entry, release, and retrograde signaling to the DHPR

Bidirectional signaling between the sarcolemmal L-type Ca2+ channel (1,4-dihydropyridine receptor [DHPR]) and the sarcoplasmic reticulum (SR) Ca2+ release channel (type 1 ryanodine receptor [RYR1]) of skeletal muscle is essential for excitation–contraction coupling (ECC) and is a well-understood prototype of conformational coupling. Mutations in either channel alter coupling fidelity and with an added pharmacologic stimulus or stress can trigger malignant hyperthermia (MH). In this study, we measured the response of wild-type (WT), heterozygous (Het), or homozygous (Hom) RYR1-R163C knock-in mouse myotubes to maintained K+ depolarization. The new findings are: (a) For all three genotypes, Ca2+ transients decay during prolonged depolarization, and this decay is not a consequence of SR depletion or RYR1 inactivation. (b) The R163C mutation retards the decay rate with a rank order WT > Het > Hom. (c) The removal of external Ca2+ or the addition of Ca2+ entry blockers (nifedipine, SKF96365, and Ni2+) enhanced the rate of decay in all genotypes. (d) When Ca2+ entry is blocked, the decay rates are slower for Hom and Het than WT, indicating that the rate of inactivation of ECC is affected by the R163C mutation and is genotype dependent (WT > Het > Hom). (e) Reduced ECC inactivation in Het and Hom myotubes was shown directly using two identical K+ depolarizations separated by varying time intervals. These data suggest that conformational changes induced by the R163C MH mutation alter the retrograde signal that is sent from RYR1 to the DHPR, delaying the inactivation of the DHPR voltage sensor