SUMMARY To assess the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro proarrhythmia model, we evaluated the concentration dependence and sources of variability of electrophysiologic responses to 28 drugs linked to low, intermediate, and high torsades de pointes (TdP) risk categories using two commercial cell lines and standardized protocols in a blinded multisite study using multielectrode array or voltage-sensing optical approaches. Logistical and ordinal linear regression models were constructed using drug responses as predictors and TdP risk categories as outcomes. Three of seven predictors (drug-induced arrhythmia-like events and prolongation of repolarization at either maximum tested or maximal clinical exposures) categorized drugs with reasonable accuracy (area under the curve values of receiver operator curves ~0.8). hiPSC-CM line, test site, and platform had minimal influence on drug categorization. These results demonstrate the utility of hiPSCCMs to detect drug-induced proarrhythmic effects as part of the evolving Comprehensive In Vitro Proarrhythmia Assay paradigm.

Recent in vitro cardiac safety studies demonstrate the ability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to detect electrophysiologic effects of drugs. However, variability contributed by unique approaches, procedures, cell lines, and reagents across laboratories makes comparisons of results difficult, leading to uncertainty about the role of hiPSC-CMs in defining proarrhythmic risk in drug discovery and regulatory submissions. A blinded pilot study was conducted to evaluate the electrophysiologic effects of 8 well-characterized drugs on 4 cardiomyocyte lines using a standardized protocol across 3 microelectrode array platforms (18 individual studies). Drugs were selected to define assay sensitivity of prominent repolarizing currents (E-4031 for IKr, JNJ303 for IKs) and depolarizing currents (nifedipine for ICaL, mexiletine for INa) as well as drugs affecting multichannel block (flecainide, moxifloxacin, quinidine, and ranolazine). Inclusion criteria for final analysis was based on demonstrated sensitivity to IKr block (20% prolongation with E-4031) and L-type calcium current block (20% shortening with nifedipine). Despite differences in baseline characteristics across cardiomyocyte lines, multiple sites, and instrument platforms, 10 of 18 studies demonstrated adequate sensitivity to IKr block with E-4031 and ICaL block with nifedipine for inclusion in the final analysis. Concentration-dependent effects on repolarization were observed with this qualified data set consistent with known ionic mechanisms of single and multichannel blocking drugs. hiPSC-CMs can detect repolarization effects elicited by single and multichannel blocking drugs after defining pharmacologic sensitivity to IKr and ICaL block, supporting further validation efforts using hiPSC-CMs for cardiac safety studies.

The region encompassing residues 181-98 on the ␣1 subunit of the muscle-type nicotinic acetylcholine receptor forms a major determinant for the binding of ␣-neurotoxins. We have prepared an 15 N-enriched 18-amino acid peptide corresponding to the sequence in this region to facilitate structural elucidation by multidimensional NMR. Our aim was to determine the structural basis for the high affinity, stoichiometric complex formed between this cognate peptide and ␣-bungarotoxin, a long ␣-neurotoxin. Resonances in the complex were assigned through heteronuclear and homonuclear NMR experiments, and the resulting interproton distance constraints were used to generate ensemble structures of the complex. The nicotinic acetylcholine receptor (nAChR) 1 (1) has long been a prototype for ligand-gated ion channels. This receptor is involved in excitatory synaptic transmission at the neuromuscular junction and also plays an important role in the nervous system. The nAChRs are pentameric complexes composed of homologous subunits with subunits arranged around the central channel in a symmetrical manner. The muscle-type nAChR contains two ␣1 subunits and one each of the ␤1, ␥(⑀), and ␦ subunits. The ligand binding sites are situated at the ␣␥(⑀) and ␣␦ subunit interfaces. The muscle-type nAChR serves as an important model for the study of the structures and functions of related ligand-gated ion channels (for review, see Ref.

Human ether-a-go-go-related gene (hERG) encodes a rapidly activating delayed rectifier potassium channel that plays important roles in cardiac action potential repolarization. Although many drugs and compounds block hERG channels, activators of the channel have only recently been described. Three structurally diverse synthetic compounds have been reported to activate hERG channels by altering deactivation or inactivation or by unidentified mechanisms. Here, we describe a novel, naturally occurring hERG channel activator, mallotoxin (MTX). The effects of MTX on hERG channels were investigated using the patch-clamp technique. MTX increased both step and tail hERG currents with EC 50 values of 0.34 and 0.52 M, respectively. MTX leftward shifted the voltage dependence of hERG channel activation to less depolarized voltages (ϳ24 mV at 2.5 M). In addition, MTX increased hERG deactivation time constants. MTX did not change the half-maximal inactivation voltage of the hERG channel, but it reduced the slope of the voltage-dependent inactivation curve. All of these factors contribute to the enhanced activity of hERG channels. During a voltage-clamp protocol using prerecorded cardiac action potentials, 2.5 M MTX increased the total potassium ions passed through hERG channels by ϳ5-fold. In conclusion, MTX activates hERG channels through distinct mechanisms and with significantly higher potency than previously reported hERG channel activators.The human ether-a-go-go-related gene (hERG) (Warmke and Ganetzky, 1994) encodes rapidly activating delayed rectifier potassium channels that conduct the cardiac I Kr (Sanguinetti et al., 1995;Trudeau et al., 1995). Although existing in many different cell types (Warmke and Ganetzky, 1994;Farrelly et al., 2003;Sarzani et al., 2006), hERG channels are highly expressed in cardiac myocytes where they function to restore resting membrane potential after action potential generation (for a recent review, see Sanguinetti and TristaniFirouzi, 2006).Many drugs with varying structures and therapeutic targets have been found to block hERG channels, which, in turn, prolong the QT interval and cause long QT syndrome (De Ponti et al., 2000, 2002 We report for the first time that mallotoxin (MTX), a natural occurring substance, is a potent and unique hERG channel activator. MTX, extracted from a tree named Mallotus phillippinensis, was originally described as an inhibitor for protein kinase C (PKC), Ca 2ϩ /calmodulin-dependent protein kinase II and III, and elongation factor-2 kinase (Gschwendt et al., 1994a,b). Recently, MTX has been shown to potently leftward shift the conductance-voltage relationship of the large conductance voltage and Ca 2ϩ -activated K ϩ channel (Zakharov et al., 2005). When we tested MTX on hERG channels, we found that MTX increased both step and tail hERG current by leftward shifting the voltage dependence of hERG activation and slowing channel deactivation. These actions distinguish MTX as a novel naturally occurring hERG channel activator. Materials and MethodsCell ...

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The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance–voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance–voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

Slob binds to and modulates the Drosophila Slowpoke (dSlo) calcium-activated potassium channel and also recruits the ubiquitous signaling protein 14-3-3 to the channel regulatory complex. RT-PCR reveals the presence of multiple slob transcripts in Drosophila heads. The transcripts are predicted to encode proteins that we call Slob51 (kDa), Slob57, Slob65, and Slob71. Slob51 and Slob65 are splice variants that lack a motif important for the binding of 14-3-3. Previous microarray analyses demonstrated the circadian cycling of slob mRNA, and we show by quantitative PCR that more than one transcript cycles in fly heads. Using in situ hybridization, we observe differences in the expression patterns of the different transcripts. Immunohistochemistry on Drosophila heads reveals Slob71/65 protein to be enriched in the lateral neurons, in contrast to Slob57/51 protein, which is expressed most prominently in the pars intercerebralis neurons and dorsal giant interneurons. Using a heterologous expression system, we show that different Slobs bind to different extents to dSlo and 14-3-3. These data reveal an unexpected diversity of the dSlo/Slob/14-3-3 dynamic regulatory complex.

Blockade of the human ether-a-go-go-related gene (hERG) potassium channel, with a consequent possibility of QT prolongation and increased susceptibility to a characteristic polymorphic ventricular arrhythmia, torsade de pointes, is an important cause of withdrawal of drugs from the market. In the aftermath of recent drug withdrawals, regulatory agencies now require in vitro hERG screening of all pharmaceutical compounds that are targeted for human use. To minimize the potential for failure in later-stage drug development, many pharmaceutical and biotechnology companies have begun to use automated patch clamp systems with higher throughput than conventional manual patch-clamp techniques to conduct routine functional hERG screening during drug discovery and early development. We have optimized an automated patch-clamp hERG screening method for the PatchXpress 7000A system (Molecular Devices, Sunnyvale, CA) using potassium fluoride (KF) in the internal recording solution. In this study we show that (1) the biophysical and pharmacological properties of hERG current recorded with KF are similar to those with standard potassium chloride solutions, (2) use of KF significantly improves the success rate of hERG screening using PatchXpress without compromising data quality, and (3) utilization of KF can significantly increase the throughput of hERG screening with PatchXpress.