PC3 cells are an epithelial prostate tumour cell line. A range of Wnts triggers a delayed Ca2+ rise [1] in these and other cancer cell lines. Recorded in patch clamp the cells have low membrane potential [2] but exhibit an outward current activated apparently at (non-physiological) positive potentials [3]. The main candidate channel carrying this K+ current is the BK(Ca) α subunit although a KCNN4 channel is also reported [4]. Without baseline subtraction, a small ‘leak’ current is sometimes apparent in recordings. All of these currents
are modulated by Wnts as described below.

As revising an ICH regulatory guideline, S7B, for the Non-clinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation by Human Pharmaceuticals) was suggested, Comprehensive In
vitro Proarrhythmia Assay (CiPA) activities are being implemented. The suggested S7B revision includes an assay of cardiac ion channels other than hERG, and CiPA-recommended voltage protocols for patch clamping was recently announced. We, Group 5 of the JSPS iSmart (investigation of in silico/in vitro model for arrhythmogenic risk prediction) play a role of wet-data collection. This time we have investigated the applicability of those protocols

In ion channel safety assessment and drug discovery it is often important to determine the mode of action of a drug candidate, which requires high-quality recordings. We successfully use the QPatch X in multi-hole mode in a screening scenario on Na_v1.5. This correlates with data from Langendorff perfused rabbit hearts, and an in vivo ECG assay using anesthetized rabbit, and effectively discriminates different modes of action of compounds on NaV1.5.
The protocol for QPatch X both tests the decay of the sodium current (30 Hz pulsetrain), and the recovery of the current. The QPatch assay allows combination of these elements into a single protocol, thereby shortening experiment time and costs.
The results show that: 1) Flecainide is a use-dependent blocker of open channels, as demonstrated by a slow decay of I_Na (30 Hz pulsetrain), and delayed recovery from inactivation in the QPatch experiments. Flecainide produces ECG changes (marked PR and QRS prolongation, and slight QTc prolongation), and lethal arrhythmia (VT ~Vf) in both Langendorff hearts and anesthetized rabbits at low doses. 2) Lidocaine is a state-dependent blocker of inactivated channels, inducing fast decay of I_Na (30 Hz pulsetrain) and fast recovery from inactivation. Lidocaine produces slight ECG changes (PR and QRS prolongation), but no arrhythmia in Langendorff-hearts or anesthetized rabbits. 3) Quinidine demonstrates a slow decay of I_Na (30 Hz pulsetrain), and delayed recovery from inactivation. Quinidine shows moderate PR and QRS prolongation, and severe morphological changes in ECGs.
The present results suggest that: 1) The QPatch X offers a time- and cost-effective screening scenario to select safe compounds with Na_v1.5 blocking activity and no proarrhythmic activity, and 2) the pro-arrhythmic activity of the Na_v1.5 blockers flecainide and quinidine might be attributable to their marked delay of recovery from inactivation.

Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs.

True giga seal patch clamping can be performed in parallel with QPatch multi-hole technology. This new multi-hole functionality has been tested on three different transient receptor potential ion channels (TRPA1, TRPV1 & TRPM8). All three targets were tested in both single-hole mode and in multi-hole mode with different agonists and antagonists. The advantages of testing compounds in multihole mode will be a minimization of biological variance and an increase in current amplitude, since the current response from several cells are summarized. The disadvantage is lack of serial resistance compensation and in some cases large leak currents

TMEM16A (ANO1) is a Ca²⁺-activated Cl- channel (CaCC) that is involved in a plethora of physiological and pathophysiological conditions. The channel was suggested as a target for treatment of asthma, secretory diarrheas, and hypertension. TMEM16A is unique as its gating synergistically depends on voltage and cytosolic Ca²⁺ (Scudieri et al. 2012).
A robust and high-throughput electrophysiology assay for TMEM16A was long awaited, but progress was hampered by the fact that many automated patch clamp devices rely on the use of fluoride (F^–)in the internal solution to promote seal formation. However CaF₂ has very low solubility and the resulting precipitation limits the ability to control precisely the concentration of Ca⁺ in the internal solution.
We recently developed a novel approach to test the pharmacological inhibition of TMEM16A on Qube with following characteristics:

• Consistently high success rates (>80%)
• Low run down
• High degree of pharmacological reproducibility (consistent IC50 values of multiple runs)

The human ether-à-go-go related gene (hERG) function is important for cardiac repolarization and inhibition of the channel can prolong the cardiac action potential, which gives increased risk for ventricular arrhythmias including torsade des points (TdP). Therefore, in vitro evaluations of the compound effects is performed on the hERG channel routinely in drug development projects to detect potential arrhythmic side-effects.
Usually these compound measurements are carried out at ambient temperatures. Previously it has been shown that the potency for many compounds has been underestimated when compared to near physiological temperature tests. Therefore, a temperature controlled measuring environment is beneficial when testing compounds for the aims as mentioned here.
Until recently, the only possibility to test compound potency under voltage control conditions has been the manual patch clamp technique. Now automated patch clamp instruments with temperature control have become available making it possible to perform up to 384 parallel recordings at controlled temperatures ranging from 8°C and above.
Here we used an automated patch clamp system, Qube, to study the effect of temperature on concentration response relationships on a panel of compounds known to block the hERG channel. Qube has a temperature controlled test environment and in these studies, we show that temperature merits being taken into consideration when evaluating for hERG pharmacology.

Several types of drug-resistant epilepsies of infancy have been associated with mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel subunit KNa1.1. Approximately forty different amino acid substitutions have been identified in patients, involving both transmembrane and intracellular domains of the channel subunit, and with one exception all cause gain-of-function in channel gating. Channel inhibition, therefore, is potentially a stratified approach to treat the disorder. To date, quinidine therapy has been trialled with several patients, but mostly with unsuccessful outcomes, which have been linked to its low potency and lack of specificity; effects on cardiac ion channels being a major concern. Here we describe the use of high-resolution structures of KNa1.1 channels, generated through cryo-electron microscopy, to identify novel inhibitors using computational methods. From an initial analysis of seventeen compounds identified using this approach, we found six that inhibited KNa1.1 channels. Their IC50 values ranged between 0.6 and 7.4 μM, compared to 100 μM with quinidine, and likely inhibited through pore-blocking mechanisms. The compounds delivered varying results when evaluating state-dependence and in preliminary toxicity assays, with some demonstrating negligible cellular toxicity and low levels of hERG block. These compounds may provide starting points for the development of novel pharmacophores for KNa1.1 inhibitors, with the view to treating KCNT1-associated epilepsies. Furthermore, this illustrates the potential for utilising ion channel structures generated through cryo-electron microscopy in drug discovery.

High information-content screenings based on whole-cell current patch-clamp recordings have become available for ion channel pharmacological research with the development of the QPatch automated patch-clamp technology. A significant increase in system throughput was recently achieved by a tripling of the number of parallel operating patch-clamp sites (from 16 to 48) by the introduction of QPatch HT. In a series of hERG screening studies we have subsequently aimed at increasing the throughput further by reducing the average experimental time consumption associated with each IC₅₀ determination, and by employing alternative calculational algorithms. We have investigated the effect of the following three procedures on effective QPatch throughput:

1. Application of multiple drugs per cell
2. Reduction of the duration of the experimental
   protocol
3. Estimation of drug potency (IC₅₀) based on a
   single inhibitor concentration

Drug discovery on ion channels is a slow and complicated process and demands a high throughput system with high data quality, but also with a flexible design and easy-to-analyze data. Sophion Qube is a next-generation giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds with a single click on a button. Data analysis is as important as data acquisition. In HTS the need for powerful analysis with efficient quality filtering is evident in order to handle the vast amount of data generated on an electrophysiological platform.

Qube is capable of testing up to 16 different cell lines or cell clones in one experiment. This can be utilized to test a different panel of cell lines or for selecting the best suited cell clone before embarking on a HTS campaign. The integrated analysis software, Sophion Analyzer, ensures analysis to keep track of all the results and whenever another QChip is assayed the analysis is done with the same set of user defined criteria.
Here we demonstrate the power of automated analysis by exploring three types of experiments executed on eight different Nav channel subtypes; 1) TTX sensitivity, 2) IV-relationship for activation and inactivation and 3) pulse train suitable for screening for use dependent sodium channels blockers. For every run Nav1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 and 1.8 were tested in parallel on a QChip. Recorded ion channel whole-cell currents were automatically analyzed for IV-relationships for activation and inactivation (V½ and Boltzmann fit and time constants) and concentration-dependent drug effects (Hill fit and IC50) were analyzed using the Sophion Analyzer. If preferred, data can by a click be exported and calculated by other programs as Spotfire, Genedata screener or implemented in in-house software.
For each subtype, the experiments identified the expected pharmacology for use- and state-dependent drugs as well as biophysical properties. The findings determined the differences between the different subtypes as expected and also that post experiment analysis can be performed with minimum of effort when using integrated, automated analysis software.

High throughput in vitro profile of the cardiac Na_V1.5 peak sodium current (I_Na) is widely used in cardiac safety screening. However, there is no standardized high throughput method to measure late I_Na. A variety of differing protocols used across industry and academia may contribute to variation in the data.
The objectives of this study were to (1) assess pharmacology and biophysical properties of veratridine- and ATX-II-induced late INa together with that induced by a mutation in the channel (ΔKPQ-Na_V1.5), (2) develop a protocol to allow simultaneous measurement of both peak and late INa under a single protocol using automated QPatch system. The planar patch clamp technique (QPatch) was applied to record the peak and late I_Na from the Na_V1.5 channel (human SCN5A gene) or ΔKPQ-Na_V1.5 mutant channel expressed in mammalian cells.
When measured at the maximal response during the ramp of the voltage protocol, the ΔKPQ-Na_V1.5 mutant produced a small late I_Na (41.9 ± 5.4 pA). Veratridine and ATX-II-induced concentration-dependent increases in the late INa. The amplitude of late I_Na were 1162.2 ± 258.5 pA and 392.4 ± 71.3 pA in the presences of 100 μM veratridine and 100 nM ATX-II, respectively. Veratridine inhibited the peak I_Na
(IC50 = 84.1 ± 10.5 μM) and altered the biophysical properties of the INa. ATX-II showed minimal effects on the peak I_Na and preserved the biophysical properties of the I_Na. In the presence of 100 nM ATX-II, potencies of 25 clinical I_Na blockers on peak and late INa were characterized. In addition, the IC₅₀ values of these clinical INa blockers on peak I_Na correlated well with and without ATX-II. The results also demonstrated that the potency of a compound blocking late I_Na could be either overestimated or underestimated if the late I_Na was measured at the end of the depolarizing pulse versus during the ramp.
In conclusion, in the presence of ATX-II, both peak and late I_Na could be assessed simultaneously under a single protocol. Our results suggest that late I_Na may be best assessed using the maximum response obtained during the ramp after 200 ms depolarizing pulse at 40 mV.

With the ever-increasing emphasis on ion channel profiling in safety screening, there has been a drive towards increased throughput with respect to patch clamp profiling. The advent of automated 384 well plate-based planar patch clamp instruments such as the Qube^® has significantly aided in this effort, allowing for a significant increase in the number of compounds that can be profiled in a time and cost-effective manner.

To further explore the possibility of increasing efficiencies in these systems we examined whether it would be possible to measure multiple currents from separate cell lines in a single well of a 384 well plate using the Qube^® instrument.

Theoretically, the instrument lends itself to this possibility due to the virtue of being able to select plates that contain multiple holes per well. Hence by adding two independent cell lines, each independently stably expressing a recombinant ion channel, it should be possible to patch two or more cell lines per well.

We have compared a variety of conditions in the study in order to generate a robust assay that allows for the simultaneous profiling of both the Na_V1.5 peak and hERG currents. Na_V1.5 and hERG currents were separated temporally by virtue of the design of the voltage protocol. Our results show that the success rates of simultaneously recording both Na_V1.5 and hERG currents in each well were 34.4%, 87.5%, 93.8%, 93.8% and 93.8% in 2, 6, 10, 16 and 36 hole/well QChips, respectively. The potencies of 28 CiPA compounds on Na_V1.5
peak and hERG currents were determined individually in a single cell line format or in a mixed cell format using a 10 hole/well QChip. The correlation of the IC50 for the compounds was excellent for both hERG (R2=0.96) and Na_V1.5 (R2=0.86) when comparing the single cell line format with the mixed cell format.

Our data demonstrate that is possible to generate a robust and reproducible assay using a single voltage protocol and buffer combination that allows for the measurement of the effects of compounds on both hERG and Na_V1.5 currents simultaneously. Such a format can both significantly increase assay throughput and reduce assay costs while maintaining data quality.

Kv1.5

Cysteine-knot miniproteins (knottins) have potential as therapeutic agents to block ion channels involved in cancer, autoimmunity and pain but suffer from manufacturing difficulties, short half-lives and a lack of specificity. IONTAS have invented a novel molecular format wherein a peripheral CDR loop (e.g. VL CDR2) of an antibody has been removed and replaced by a naturally occurring knottin. In this novel format (termed a KnotBodyTM), the knottin enjoys the extended half-life of an antibody molecule and the peripheral CDRs gain additional diversity within a scaffold which is pre-disposed to blockade of ion channels. This example of successful fusion of one structural domain within another was initially achieved by inserting a trypsin binding knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBodies were selected from this library using phage display technology on the basis of retained trypsin binding and the correct folding of both domains were confirmed using X-ray crystallography. To further demonstrate the merits of this novel format, the modular nature of the KnotBody binding surface was exploited to: (i) improve existing knottin binding by introducing additional VHcontacts; (ii) create a bispecific molecule by introducing a VH chain that binds to a different target; (iii) engineer novel binding specificity on the knottin scaffold by loop diversification; (iv) substitute the selected (EETI-II trypsin binding) knottin with ion channel blocking knottins.

The MaxCyte® STX™ Scalable Transfection System uses a proprietary flow electroporation technology that can transfect up to 1E10 cells with target, reporter and protein expression plasmids, as well as other molecules, in less than 30 minutes. Transfected cells can be assayed immediately or cryopreserved for future use. Here we demonstrate the use of the MaxCyte STX system in coordination with Corning’s ® HYPERFlask® Cell Culture Vessel to provide an efficient and economical solution for culturing large numbers of adherent CHO cells before and after transfection. The HYPERFlask Cell Culture Vessel features Corning’s HYPER (High Yield PERformance) technology, which utilizes a gas permeable film to provide gas exchange between the internal culture environment and the external atmospheric environment. The unique, space-saving, 10-layer film design results in 1720 cm2 cell growth surface area, which is approximately 10 times that of a normal T-175 flask. We also show that cells transfected with a plasmid encoding a Kv1.3-GFP fusion protein exhibited good seal formation and strong potassium currents following large scale electroporation and cryopreservation. Ion channel activity was assayed on the Sophion QPatch automated patch clamp system. High viability and consistent levels of expression were obtained in three independent large scale electroporations, illustrating robustness and reproducibility of the transfection process. These results demonstrate that the MaxCyte STX system offers a time and labor saving alternative to stable cell line generation for ion channel assays.

The Comprehensive in vitro Proarrhythmia Assay (CiPA) proposal provides an attractive perspective for increasing the efficacy of the drug development process. A detailed electrophysiological analysis of Na_v1.5 (peak and late currents), K_v4.3 (I_to), hERG (I_Kr), KvLQT1/minK (I_Ks), Ca_v1.2 and Kir2.1 (IK1) upon addition of a potential drug is a major part of assessing the drug’s proarrhythmic risk. Gaining a better understanding of the complex relationship between QT-elongation and the occurence of Torsades des Pointes (TdP), potentially enables compounds with properties that today are considered as problematic to be further developed. High throughput screening (HTS) supports this quest and is furthermore of great importance for discovering pharmacologically active substances and understanding ion channels.

Qube is a giga-seal automated patch clamp (APC) instrument, providing 384 amplifiers for the consumable QChip 384 with its integrated electrodes. The QChip has built-in microfluidic flow channels that ensure a fast and complete exchange of liquid for reliable measurements on ligand-gated ion channels and sequential additions to the same site. Here, four different cell lines expressing the cardiac ion channels Na_v1.5, Ca_v1.2, K_v1.5 and hERG, were transferred from a cell clone cell transfer plate (ccCTP) onto the same QChip. A range of pharmacological substances and voltage step protocols were applied to address the suitability of Qube for measuring different cell populations in parallel.

The Qube is the new gigaseal-based 384-channel planar patch clamp system developed by Sophion Bioscience A/S. The system is equipped with 384 individual amplifiers providing continuous recordings at a sampling rate up to 50 kHz from all wells simultaneously. The consumable for the Qube is built on Sophion’s tried and tested silicon technology for optimal giga-seal formation and has been designed to have an effi cient liquid fl ow system which is necessary for testing fast ligand-gated ion channels (LGIC) such as the nicotine acetylcholine receptors (nAChR). In this work we present recordings obtained using the Qube system from two LGICs: ASIC1a and nAChRα1.
The acid-sensitive channel (ASIC) belongs to the ENaC/DEG family. ASIC1a is expressed in the brain and in the peripheral nervous system where it is involved in modulating response to pain (1). The nicotinic acetylcholine receptor alpha 1 (nAChRα1) belongs to the Cys-loop family and is expressed both centrally and in the peripheral nervous system. In the periphery nAChRα1 modulate e.g. the synaptic transmission at the neuromuscular junction (2). Here we show activation of these LGICs by their respective ligand (H+ and acethylcholine (ACh), respectively) and pharmacological block by known reference compounds (amiloride and tetracaine, respectively). These data demonstrate: 1) the design of the Qube consumable allows fast liquid exchange for recordings of fast LGICs and 2) the design of the Qube consumable allows multiple liquid additions to the same recording unit.

Drug discovery on ion channel targets has been an under-explored territory because suitable screening tools for patch clamp were missing. This changed during the 2000’s with the introduction of automated patch clamp (APC) devices. Still, the throughput and running cost of APC devices did not allow for the employment of the patch clamp assays in primary screening. Because of this bottleneck, APC has remained a secondary screening tool. We are now presenting data from the first true gigaseal-based 384-channel planar patch clamp system, the Qube, capable of providing the throughput needed for primary screening. The Qube is a collection of years of experience with planar patch clamp devices: High quality, reliability and easy to use concepts are the core in both the tried-and-tested silicon technology of the consumable, and in the technologies used in the instrument. The Qube offers GΩ seals and efficient integrated liquid flow in 384-well format.
We show here that the Qube provides exceptional data quality for electrophysiological assays with fast desensitizing ligand-gated ion channels. The fast liquid exchange permits accurate XC50 estimations. We have tested the acid sensing ion channel (ASIC1) and the nicotinic acetylcholine receptor (nAchRα1) on the Qube in both agonist and antagonist configurations, showcasing the instrument’s broad flexibility in terms of assay design. The results prove that: 1) Repetitive agonist applications result in reproducible current responses. 2) The agonist can be applied and washed off an unlimited number of times as long as the cell is stable. 3) XC50 values correspond to expected values (QPatch and literature). Collectively, our results show that the design of the flow channels on the Qube consumable enables recordings on ligand-gated ion channels with the highest throughput capacity seen so far, without compromising data quality, reproducibility or reliability.

A series of dihydropyrazole derivatives was developed as potent, selective, and brain-penetrating T-type calcium channel blockers. An optimized derivative, compound 6c, was advanced to in vivo studies, where it demonstrated efficacy in the WAG/Rij rat model of generalized nonconvulsive, absence-like epilepsy. Compound 6c was not efficacious in the basolateral amygdala kindling rat model of temporal lobe epilepsy, and it led to prolongation of the PR interval in ECG recordings in rodents.

Recent work by FDA and HESI CiPA working groups indicate that in vitro hERG, Nav1.5 and Cav1.2 potency data in addition to dynamic hERG kinetic data is required to accurately predict proarrhythmic risk. Below we explore two key challenges in exclusively using automated patch clamp for risk prediction:

1. Metrion has previously shown the ability to implement the difficult Milnes protocol on QPatch but the challenges of producing full concentration-response formats required for in silico models are unknown
2. Cav1.2 inhibition values for compounds such as verapamil have been
   variable, with literature IC50 values >10 µM compared to the 202 nM IC50 obtained by manual patch clamp in the original CiPA study. The similar potency of verapamil against Cav1.2 and hERG make it a Multiple Ion Channel Effect (MICE) compound and “rescues” it from being classed as a high-risk proarrhythmia compound as shown by Li et al.
3. QPatch dynamic hERG and Cav1.2 data from multiple voltage protocols
   were used to assess proarrhythmic risk using the FDA’s in silico model.

We here report pharmacological data (EC₅₀ and IC₅₀ values, tests for state-dependency of blockers and steady-state inactivation characteristics) from eight different types of voltage- and ligand-gated ion channels using the automated QPatch 16 system. The data obtained were in accordance with published literature values (not shown). We conclude that the QPatch technology enables fast and reliable characterization of electrophysiological ion channel properties.

The human α7 nicotinic acetylcholine receptor (α7-nAChR) is a neuronal ligand-gated, fast desensitizing, non-selective cation channel. It is pentahomomeric, consisting of five 50 kD α7 subunits, each composed of 502 amino acids. The α7-nAChR is involved in memory and cognition, and it is widely distributed throughout the nervous system, especially in cholinergic neurons projecting to hippocampus and cortex. The α7-nAChR has also been found to be associated with pathophysiological states. Importantly, it is involved in widespread human neuro-degenerative and psychotic disorders, including Alzheimer’s disease and schizophrenia. Therefore the therapeutic potential of α7-nAChR is substantial, and electrophysiological and pharmacological characterization of the receptor has become an increasingly important issue. The fast kinetics (milliseconds) makes precise patch clamp measurements a challenging task. The present study addresses the possibility of efficient and precise characterization of α7-nAChR function and pharmacology by means of the QPatch automated patch clamp system. Compared to conventional whole-cell patch clamp the system enables a highly increased throughput by simultaneous and asynchronous operation of 16 parallel patch clamp experiments.

Cultured cells from mammalian cell lines incubated at 37 °C can be used for automated patch clamp (APC), including compound screening, for only a relatively brief period of time. Usually this time period extends from the time of ~50 % confluence to full confluence. For CHO cells, the preferred cell type for APC, expressing hERG potassium channels the duration of the usable period is 1-2 days. We here report that incubating CHO-hERG cells at a reduced temperature, 30 °C, for 1-5 days prior to APC experiments is advantageous in several ways: (1) it increases the percentage of cells with acceptable hERG currents (> 50 pA), (2) it increases the average whole-cell current significantly, and (3) it
slows cell proliferation and extends the usable period up to at least 5 days. In addition, we have tested whether the altered conditions affect the IC50 obtained for known hERG inhibitors. For all experiments the automated patch clamp system QPatch 16 was used.

Drug-induced QT interval prolongation, ventricular arrhythmia and sudden death are one of the leading causes for drug withdrawal from the market or denied regulatory approval. Preclinical profiling for off-target cardiac ion channel interactions using The Comprehensive in vitro Proarrhythmia
Assay (CiPA) paradigm has been initiated to overcome drug attrition due to hERG inhibition only. The aim is to improve the prediction of a drug’s proarrhythmic liability for the pharmaceutical industry. This new paradigm includes a panel of in vitro assays that integrates effects of the test compounds on several cardiac ion channels. Voltage-gated ion channels in this panel, particularly hCa_V1.2 and hKCNQ1/hminK, exhibit rapid
activities decrease when contact between membrane and cytosol is disrupted upon patch excision (run-down). The loss of channel activity without pharmacological interaction poses a challenge for developing accurate, precise and robust assay yielding high success rate, minimal current run-down and reliable pharmacological results. In the present study, hCa_V1.2 and hKCNQ1/hminK cardiac ionic currents were validated on QPatch HT patch clamp system with the specific focus on preventing current run-down. The results demonstrate suitability of these assays for screening and profiling of drug effects by significantly reducing run-down (<20%) by optimizing protocol parameters. Reference pharmacology was
assessed for Ca_V1.2 (Nifedipine IC₅₀ =144nM) and hKCNQ1/hminK (Chromanol 293B IC₅₀ =12.3μM). The optimized assays for hCa_V1.2 and hKCNQ1/hminK on the QPatch are able to provide data that is comparable to manual patch clamp, which allows the assessment of a novel compounds proarrhythmic risk.

Optical modulation of ion channels is traditionally studied using a manual patch clamp system combined with a light source. This approach, however, is limited by very low throughput. In the present work, we show data recorded using a 384-well based automated patch clamp system equipped with 384 integrated light sources (Qube Opto 384).

Optogenetics
In this study, we evaluated Channelrhodopsin 2 (ChR2), a light-sensitive non-selective cation channel permeable to Na+, K+ and Ca2+ opened upon illumination (Berndt et. al., 2012). Furthermore, we employed the chloride-conducting channelrhodopsin (iC++, Govorunova et Al. 2015), which was developed from a non-selective cation conducting channelrhodopsin through a mutational approach. Finally, we used photoactivatable adenylyl cyclase, bPAC, to modulate cellular cAMP levels and thereby alter biophysical properties of the HCN2 channel (Stierl et Al., 2011).

Optopharmacology
Compound activation by light enables the pharmacological manipulation of receptors, ion channels and other proteins with a high degree of temporal control. We used caged GABA (Rubi-GABA, Zayat et. al., 2003) to study the light activation of ligands, in combination with the microfluidic flow channel of the QChip 384, to give a higher degree of experimental control.

Optical modulation of ion channels is traditionally studied using a manual patch clamp system combined with a light source. This approach, however, is limited by a very low throughput. In the present work, we show data recorded using a 384-well based automated patch clamp system equipped with 384 integrated light sources (Qube Opto 384).

Optogenetics
In this study we evaluated Channelrhodopsin 2 (ChR2), a light-sensitive non-selective cation channel permeable to Na⁺, K⁺ and Ca²⁺ opened upon illumination (Berndt et al., 2012). Furthermore, we employed the chloride-conducting channelrhodopsin (iC++, Govorunova et al., 2015), which was developed from a non-selective cation conducting channelrhodopsin through a mutational approach.

Optopharmacology
Compound activation by light enables the pharmacological manipulation of receptors, ion channels and other proteins with a high degree of temporal control. We used caged GABA (Rubi-GABA, Zayat et al., 2003) to study the light activation of ligands, in combination with the microfluidic flow channel of the QChip 384, to give a higher degree of experimental control.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors permeable to Ca²⁺, Na⁺ and K⁺. To be activated, they need to bind to glutamate (via GluN2 subunits), glycine (via GluN1) and release the Mg²⁺ blockade by
membrane depolarization. The majority of NMDARs are tetrameric complexes, consisting of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. GluN1 is coded by a single gene with at least eight different splice variants; four different GluN2 genes originate GluN2A, GluN2B, GluN2C, and GluN2D subunits. NMDARs containing different GluN2 subunits have different pharmacological and kinetic properties.

NMDA receptor modulators can be studied using a variety of techniques.

New cardiac safety testing guidelines are being developed as part of the FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, which aims to remove the reliance on screening against the hERG channel by expanding the panel to include other human ventricular ion channels such as Nav1.5, Ca_V1.2, K_V4.3/KChiP2.2, K_ir2.1 and K_V7.1/KCNE1. In addition, the CiPA working groups have recently identified two additional ion channel assay readouts required for in silico models to reliably predict proarrhythmia. The first is a ‘late’ Na_V1.5 assay, as inhibition of persistent inward current can affect repolarisation and mitigate proarrhythmia (e.g. Ranolazine). The second is a kinetic hERG assay that measures drug trapping using the Milnes voltage protocol(1) and improves the prediction of proarrhythmia risk(2). Here we describe validation of these additional CiPA assays on the gigaseal QPatch48 automated patch clamp platform.

Torsades de pointes (TdP) is a potentially fatal type of a ventricular tachycardia associated with delayed repolarization of the cardiac action potential. The major reason for pharmacologically induced TdP is the blockade of the voltage-gated potassium channel (KV11.1 or hERG current, I_Kr).
Therefore, the main focus of pre-clinical in vitro tests has been set on detection of I_Kr blockade to effectively discard drugs with a propensity to induce TdP. However, not all compounds that block I_Kr will eventually induce tachyarrhythmia and, therefore, a detected block of I_Kr alone is not specifically
predictive for delayed repolarization and TdP. Not all known I_Kr blockers cause significant arrhythmia because effects caused by induced reduction of potassium outward currents may be
counterbalanced by a reduced calcium inward current (ICaL) or late inward sodium current (I_NaL). hERGand L-type calcium currents (Ca_V1.2) can easily be assessed in in-vitro systems using electrophysiology methods (e.g. patch-clamping). Since the physiological late sodium current exhibits only tiny current amplitudes, I_NaL needs to be increased for drug screening by decreasing or slowing the inactivation of Na_V1.5 channels. This can be pharmacologically achieved by adding a sea anemone toxin II (ATX-II), which binds to the extracellular linker of segments S3-S4 of domain IV or by using inactivation modifying mutations.

For this study, a cell line stably expressing a mutated NaV1.5 (CW) channel was generated and validated using known I_NaL blockers. The substitution L409C/A410W was found to lead to an inactivation-deficient mutation. The mutation is localized in D1S6 and presumably prevents access of the intrinsic fast inactivation particle to the inner cavity. During pharmacological validation using manual and automated (QPatch™) patch-clamping, IC₅₀ values differed by less than a factor of two between ATXII stimulated and CW mutated Na_V1.5 channels. Besides shorter duration of I_NaL experiments and larger current amplitudes, also the observed conserved sensitivity to ATXII and overall reduced assay costs are strong arguments to
screen late sodium currents in mutated rather than in pharmacologically stimulated Na_V1.5 channels.

High-throughput automated patch clamp (APC) is quickly emerging as the new gold standard technology for screening compounds against ion channel targets. The latest APC platforms, such as the Qube, enable true giga-seal quality data generation, comparable to conventional patch clamp.

Traditional plate-based screening technologies rarely allowed for analysis of multiple parameters to detect potential differences in the mechanism of action of novel compounds. Using multiple data analysis cursors in the Sophion Analyzer software, it’s possible to gain valuable insight into compound activities on a particular ion channel target, which may translate into different results in further in vivo experiments. This also increases the value of APC data, as multiple parameters can be extracted from each well for both data quality control as well as hit detection.

Here we present Qube data for a 10,000 compound pilot screen for a sodium channel activator project, with analysis for multiple hit parameters, demonstrating the ability of the assay to detect novel compounds with different mechanisms of action.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS) and the binding of GABA to ionotropic GABA receptors (GABA_AR) is a crucial process in the healthy brain. An imbalance of GABA secretion or the malfunction of the receptor is associated with multiple disease areas like anxiety disorders, seizures and schizophrenia. Pharmacological manipulation of the receptor has, therefore, a large therapeutic potential, which is underscored by the amount of available treatment possibilities and the ongoing search for alternatives thereof^1-4.
GABA_A receptors are ligand-gated ion-channels that consist of 5 membrane-spanning subunits^5,6 and are permeable to Cl^– ions. So far, 16 different subunits have been identified in humans (ɑ1-6, ꞵ1-3, γ1-3, δ, ε, γ, θ, π) and their composition within the GABA receptor leads to different pharmacological responses7. Here, we show pharmacological modulation of the GABA_AR using our high-throughput automated patch clamp (APC) systems QPatch and Qube 384. Our study includes a characterization of the heterogeneous GABA_AR population of cultured primary hippocampal astrocytes and an evaluation of the GABA_AR clone ɑ5ꞵ3γ2. In addition, we utilize the well-characterized GABA_AR response to establish a novel method for ligand release, namely the light-stimulated release of ruthenium-bipyridine-triphenylphosphine(RuBi)-caged GABA using light stimulation on the Qube 384 platform.

The ongoing pain associated with arthritis reduces the quality of human life. Injection of complete Freund’s adjuvant (CFA) into the knee of mice induced inflammation and a concomitant decrease in their natural digging behaviour within 24-hours. After inflammation, retrogradely labelled knee-innervating dorsal root ganglion neurons showed a decreased action potential threshold and increased transient receptor potential vanilloid-1 (TRPV1) expression (validated with immunohistochemistry). Subsequently, systemic administration of a TRPV1 antagonist normalized mouse digging behaviour within 30-minutes, likely due to inhibition of pain signalling. To test the translational potential of these results, we recorded from mouse sensory neurons incubated overnight with synovial fluid from humans with osteoarthritis experiencing chronic pain (OA-SF). We find that OA-SF directly sensitizes mouse knee neurons by decreasing the action potential threshold and increasing the resting membrane potential implicating voltage-gated sodium (Na_v) and potassium (K_v) channels. Using Ca²⁺-imaging we also find that OA-SF increases TRPV1 and TRPM8 activity compared to synovial fluid from healthy individuals, highlighting the importance of TRP channels in OA pain. Taken together, our data suggest that inflammatory mediators in OA-SF excite sensory neurons and hence drive knee pain. This in vitro, translational model can be incorporated into clinical trials to unbiasedly assess the efficacy of drugs on sensory neurons and hence identify drivers of OA pain.

Growing functional and genetic evidence points to an emerging role for two-pore domain potassium channels (K2Ps) in numerous pathophysiologies. K2Ps carry background (or leak) potassium current and primarily function to maintain cellular resting membrane potential. However, K2Ps have proved a challenging superfamily to modulate with small molecules and there is currently a lack of pharmacological tools which target K2Ps. This has limited the investigation of K2Ps precise physiological function and efforts to generate therapeutics which act upon K2Ps. Our initial aim was to develop a screening system which would allow the identification of small molecule K2P activators. We initially showed that the identification of channel activators can be compromised in systems in which the target (K2P) is over-expressed at high levels. To avoid this, we used baculovirus (‘BacMam’) to transduce K2P expression in mammalian cells. ‘BacMam’ allows the precise titration of expression of the gene of interest. This enabled us to develop cell systems in which we were able to intricately and robustly select a level of K2P expression (via functional studies) optimized for the identification of channel activators. An example assay development using the K2P TRAAK is described. TRAAK is known to be expressed in nociceptors and has been shown to be a genetic predictor of post-surgical neuropathic pain. An appropriate level of TRAAK channel function was chosen after ‘BacMam’ optimization and channel function measured via thallium flux. The pharmacology of previously described TRAAK activators is also shown. A representative sub-group of K2Ps (TWIK-1, THIK-1, TASK-3, TASK-2, TREK-2) was screened using the BacMam/ Thallium flux assay against the LifeArc Index set (11k compounds). This allowed us to assess which K2Ps were ‘ligandable’ or ‘activatable’ by small molecules. Multiple novel activators were identified, and development is on-going. We also screened a library of drug-like molecules against the same group of K2Ps and identified Terbinafine as a novel activator of the TASK-3 channel. The selectivity of this molecule across the K2P superfamily was assessed and structure-activity relationship studies showed that relatively small changes to the molecule had significant effects on activity and selectivity.

The voltage-gated sodium channel, Na_V1.1, highly expressed in fast-spiking interneurons (FSIs), initiates the interneuron’s action potentials and promotes γ-oscillations. This process is critical for memory encoding and other cognitive functions. An impaired function of FSIs is associated with disorders like autism, schizophrenia, Alzheimer’s disease and others. Potentiators of Na_V1.1 can restore γ-oscillations by recovering reduced FSI function. These compounds were further shown to mitigate cognitive dysfunction in transgenic mice with decreased levels of Na_V1.1 expression in parvalbumin-positive neurons.

Na_V1.1 gating is complex, and activators of the ion channel can modulate various channel properties. Amongst many different screening techniques available, the patch clamp technique is the only one that reveals mode of action information. Recent advances of patch clamp devices have enabled the technology to be employed in large compound library screens. Qube 384 is a fully automated 384-well patch clamp device capable of testing thousands of compounds per day whilst providing true giga-ohm seal quality data. Using the Qube 384 in a drug discovery cascade enables acquisition of mode of action data simultaneous with hit detection during the primary screen, minimizing the need for many follow-up validation studies.

Introduction
Human induced pluripotent stem cells (hiPSCs) can be differentiated into many cell types, including neurons and cardiomyocytes, and therefore constitute a novel way to model human diseases for drug testing in vitro1. Ion channels represent attractive therapeutic targets in the nervous and cardiovascular systems2 rendering electrophysiological studies of hiPSCs interesting for their usage in drug discovery. However, such studies have traditionally been limited by the labour-intensive and low throughput nature of patch clamp electrophysiology2.

Here we present the electrophysiological characterization of hiPSC-derived motor neurons using our automated patch clamp (APC) platforms, Qube 384 and QPatch. Our results include a measure of channel expression versus time in culture, the pharmacological dissection of endogenous ion channels (e.g. voltage-gated Na+ (NaV) and voltage-gated K+ (KV) channels), identification of ligand-gated receptors and recordings of action potentials using current clamp. The major challenge when investigating neurons using APC platforms is the requirement to dissociate the cells from their neuronal network while maintaining cell viability and membrane integrity3. By optimization of the harvest- and whole-cell protocols we have overcome this obstacle resulting in success rates of up to 60% using our 384-well APC system. Utilizing the high throughput nature of our system we tested in parallel two disease models, using hiPSC neurons derived from Spinal Muscular Atrophy (SMA) or Amyotrophic Lateral Sclerosis (ALS) patients, together with control cells from healthy subjects and isogenic control cells. The results show a general overactivity of Na+ channels in both cell lines, which could be rescued by a point mutation in the superoxide dismutase (SOD1) gene in the isogenic ALS cell line.

Our results demonstrate the feasibility of conducting electrophysiological characterization and screening of hiPSC-derived neurons on APC platforms like Qube 384 and QPatch, thus paving the way for high throughput ion channel-targeted screening of drugs for neurological disorders.

Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels. A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion channel targets accessible for drug screening. Specifically, genuine HTS parallel processing techniques based on arrays of planar silicon chips are being developed, but also lower throughput sequential techniques may be of value in compound screening, lead optimization, and safety screening. The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery.

The citrus flavanone hesperetin (HSP) has been proposed for the treatment of several human pathologies, but its cardiovascular actions remain unexplored. Here we studied the effects of HSP on the human cardiac voltage-gated Na+ channel (hNaV1.5) and compared it to its effects on a recombinant hNaV1.5 channel baring a mutation (R1623Q) associated with lethal ventricular arrhythmias in the Long QT syndrome type 3 (LQT3).

METHODS: Whole-cell patch clamp experiments were used to record Na+-currents (INa) in HEK293T cells expressing hNaV1.5 wild type (WT) or mutant channels.

RESULTS: HSP blocked thehNaV1.5 channels in voltage-dependent manner with an IC50 ≈ 100 μM. Its inhibition was decreased by disruption of the F1760 residue. HSP preferentially accelerated the inactivation phase of INa and decreased the Na+ net influx into the cell. The effects of HSP on the inactivation phase and voltage-dependent inhibition of INa were more marked in the LQT3 mutant.

CONCLUSIONS: HSP could be used as a template to develop drugs against cardiac arrhythmias in LQT3.

Background and Purpose

TREK two‐pore‐domain potassium (K2P) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI‐530159.

Experimental Approach

The effect of GI‐530159 on TREK channels was demonstrated using 86Rb efflux assays, whole‐cell and single‐channel patch‐clamp recordings from recombinant TREK channels. The expression of K2P2.1 (TREK1), K2P10.1 (TREK2) and K2P4.1 (TRAAK) channels was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current‐clamp recordings from cultured rat DRG neurons were used to measure the effect of GI‐530159 on neuronal excitability.

Key Results

For recombinant human TREK1 channels, GI‐530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current‐clamp recordings from cultured rat DRG neurones showed that application of GI‐530159 at 1 μM resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential.

Conclusions and Implications

This study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI‐530159 could aid in the development of novel analgesic agents.

Pathogenic T cell effector memory (TEM) cells drive many autoimmune disorders and are uniquely dependent on the Kv1.3 channel. A number of venom derived knottin (cysteine-rich mini-protein) inhibitors of Kv1.3 are being developed as potential drug candidates, but can suffer from manufacturing difficulties, short half-lives and a lack of specificity. We have developed a novel molecular format wherein a peripheral CDR loop of an antibody has been replaced by a knottin. In this novel KnotBody^TM format, the knottin benefits from the improved therapeutic functionality of an antibody and the antibody gains additional diversity by the addition of a scaffold which is pre-disposed to blockade of ion channels.

A proof-of-concept fusion protein of one structural domain within another was initially achieved by inserting a trypsin inhibiting knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBody^TM molecules were selected from this library using phage display technology on the basis of retained trypsin binding, with the correct folding of both domains confirmed using X-ray crystallography.

To further demonstrate the benefits of this novel format, the modular nature of the KnotBody^TM binding surface was exploited to: (i) improve existing knottin binding by introducing additional V_H contacts; (ii) create a bispecific molecule by introducing a V_H chain that binds to a different target; (iii) substitute the proof-of-concept knottin (EETI-II, a trypsin inhibitor) with ShK, a Kv1.3 ion channel blocking toxin; (iv) develop a panel of low-nM Kv1.3 inhibitors with selectivity exceeding 3000-fold over the Kv1.1 channel, a closely related Kv family member.

The major inhibitory neurotransmitter of the central nervous system is γ-aminobutyric acid (GABA) and GABA is exerting its effect by binding to GABA receptors. The central role of GABA in the nervous system is underscored by the devastating consequences of pathophysiological changes in GABA signalling. Conversely, manipulation of GABA receptors can offer relief of a large group of neurological and psychiatric disorders. Pharmacological manipulation of GABAA has a large potential and ligands increasing the current will typically have anxiolytic, anticonvulsant, amnesic, sedative, hypnotic, euphoriant, and muscle relaxant effects.

GABAA receptors are ligand-gated ion channels, permeable to Cl- ions, consisting of 5 membrane-spanning subunits. 16 different subunits are identified in humans (α1-6, β1-3, γ1-3, δ, ε, θ, π) and the cellular GABA response is hence composed by a population of GABA receptors with significant different pharmacology. Here we demonstrate pharmacological GABA receptor evaluation in both a stably-transfected cell line containing only α5β3γ2 receptors and a primary cell culture of rat hippocampal astrocytes with a diverse GABA receptor population.

The major inhibitory neurotransmitter of the central nervous system is γ-aminobutyric acid (GABA) and GABA is exerting its effect by binding to GABA receptors. The central role of GABA in the nervous system is underscored by the devastating consequences of pathophysiological changes in GABA signalling. Conversely, manipulation of GABA receptors can offer relief of a large group of neurological and psychiatric disorders. Pharmacological manipulation of GABAA has a large potential and ligands increasing the current will typically have anxiolytic, anticonvulsant, amnesic, sedative, hypnotic, euphoriant, and muscle relaxant effects^1–4.
GABAA receptors are ligand-gated ion channels, permeable to Cl- ions, consisting of 5 membrane-spanning subunits^5,6. 16 different subunits are identified in humans (α1-6, β1-3, γ1-3, δ, ε, θ, π) and the cellular GABA response is hence composed by a population of GABA receptors with significant different pharmacology⁷. Here we demonstrate pharmacological GABA receptor evaluation in both a stably-transfected cell line containing only α5β3γ2 receptors and a primary cell culture of rat hippocampal astrocytes with a diverse GABA receptor population.

Voltage-gated sodium channels are transmembrane proteins that are responsible for the rapid depolarization that elicits the upstroke of action potentials in neurons and cardiac cells. Molecular cloning techniques have identified nine related voltage-gated sodium channels subtypes with distinct biophysical properties, interacting proteins, and cellular patterns of expression that are involved in electrical signaling. The goal of many research groups is to target specific sodium channels subtypes to develop selective inhibitors that could, in theory, produce safer and more efficacious drugs. To address compound sodium subtype profiling, we used a two-pulse
state-dependent protocol to develop a sub-type specific sodium channel (hNav1.1 to hNav1.8) panel on the QPatch HT patch clamp system. Additionally, we tested three stimulus protocols on hNav1.5 with known sodium inhibitors: 1) CiPA protocol for cardiac safety testing (tonic), 2) two-pulse state-dependent protocol (inactivation-dependent), 3) three-pulse protocol (tonic, inactivation-dependent, and V1/2 dependent inactivation). The CiPA protocol identified state-independent compounds (e.g. Flecainide) within three-fold of published
literature values but didn’t identify state-dependent compounds such as Lidocaine. The two-pulse protocol identified both state-dependent and independent compounds at pulse 2 with IC50 values within 4-fold published literature values. Data from the first two pulses of the three-pulse protocol had comparable pharmacology to the two-pulse protocol. The functional activity from V_1/2 inactivation (a delayed 3rd pulse) was less predictive of state-dependent inhibition than the two-pulse protocol but may reflect the in vivo inhibition. The hNav1.x panel using the two-pulse protocol can be useful to profile target identification, selectivity and state-dependence of novel compounds. (QPatch Automated Patch Clamp System is manufactured by Sophion Bioscience).

There is increasing interest for cardiomyocytes as models for studying cardiac cellular physiology and preclinical drug safety testing. Stem cell-derived cardiomyocytes have the potential for such a model and have the possibility for modeling human diseases. The present investigation is the first to describe current properties from stem cell-derived cardiomyocytes using multi-hole recordings with planar automated patch clamp technology. In our study pluripotent stem cell-derived cardiomyocytes were biophysically and pharmacologically characterized. The cells are differentiated in large numbers and cryo-preserved, which make them suitable for automated patch clamping and facilitate their use in drug screening. We tested the cells in two different recording modes; single-hole and multi-hole, respectively. For multi-hole recordings up to ten cells are patched at the same time and the total current is measured per site. This recording mode can be useful for small currents (e.g. endogenous) and typically increases the success rate for useful data. For all experiments the whole-cell configuration was used and three different types of currents were studied; Na⁺, Ca2⁺ and K⁺. Using specific voltage protocols biophysical characteristics of each current was described and compared from single-hole and multi-hole experiments. We showed that currents recorded from these pluripotent stem cell-derived cardiomyocytes are similar to human cardiomyocytes and the response to known pharmacology is as expected. The V0.5 values, I-V relationships, current kinetics and IC₅₀ values determined for known blockers (TTX, nifedipine and cisapride) were comparable for the two recording modes. Clearly the success rate for usable data per measurement plate was significantly increased with the multi-hole technology. This is the first time current properties of stem cell-derived cardiomyocytes have been described from multi-hole recordings with planar automated patch clamp. Our study has shown that automated patch clamp is ready for stem cell-derived exploration.

The QPatch HTX is the newest member of the of the QPatch family of high-end fully automated patch clamp systems from Sophion Biosciences. The QPatch HTX builds on the proven technology from the QPatch 16 and QPatch HT, where cells are patched on a silicon chip with integrated microfluidic flow channels made in glass. The chip, called a QPlate, allows true gigaseals to be formed and high quality data to be recorded. For QPatch HTX modifications has been made to the QPlate and to the amplifier. To diminish problems with low-expressing cell lines, the QPlate HTX contains multiple holes per recording site and to accommodate the increased current amplitudes, the amplifier has been built with a broader current range.
In this study we present data obtained during the assay validation and optimization of a high throughput screening assay using a HEK293 cell line expressing the voltage gated sodium channel Na_v1.2a. This stable cell line is characterized by a very low functional expression of the Na_v1.2a channel with approximately 30% expressing cells in the population resulting in a relatively low success rate in data recording on QPatch HT. We found that with the QPlate HTX the success rate could be dramatically improved and upon assay optimization on QPatch HTX the effective success rate approaches 100%. We present data to compare throughput and assay quality between the QPatch HTX in multi-hole mode and traditional single cell patch clamp on QPatch HT.
Furthermore we have evaluated the compound profiling properties of the QPatch HTX by characterizing known blockers against the Na_v1.2a channel – a challenging channel for electrophysiological recording due the extremely low time constants of the response elicited upon voltage stimulation of the channel. Surprisingly, our biophysical and pharmacological data suggest that the data quality obtained on the QPatch HTX system is overall comparable to equivalent data obtained on the QPatch HT system.

The human ether-à-go-go related gene (hERG) carries one of the major repolarizing currents in the heart myocytes and block of this channel by certain drugs may lead to prolongation of the cardiac action potential and the potentially lethal arrhythmia torsade de pointes (TdP). Therefore current guidelines are that potency against hERG should be assessed in preclinical drug development. However, not all compounds inhibiting hERG are proarrhythmogenic and more integrated effects of the rug must be taken into consideration.

The comprehensive in vitro proarrhythmic assay (CiPA) initiative have introduced a new paradigm for assessing
proarrhythmic risk based on a thorough understanding of the underlying cellular mechanisms leading to a more predictive estimation of the risk carried by the specific compound.

One pillar of the CiPA paradigm is to measure the major
ionic currents involved in cardiac depolarization and repolarization using the high throughput automated patch clamp (APC) systems. Until recently this has meant that the measurements were carried out at ambient temperature a but recently automated patch clamp instruments with temperature control have become available making it possible to perform up to 384 parallel recordings at controlled temperatures ranging from 10 to 40°C.

Here we estimate potencies and binding kinetics to the hERG channel which are important parameters for the outcome of the risk assessment for a panel of clinical drugs using a Qube APC system equipped with temperature control.

The QPatch HTX automated patch clamp technology was developed to 1) increase throughput in ion channel drug screening by parallel operation of 48 multi-hole patch clamp sites, each comprising 10 individual patch clamp holes, in a single measurements site on a QPlate X, and 2) diminish problems with low-expressing cell lines. Thus, parallel recording from 10 cells represents a 10-fold signal amplification, and it increases the success rate at each site substantially.
To further increase throughput we explored the possibility of simultaneous recording of a number of ion channel currents. Two or three cell lines, each expressing a specific ion channel, were applied at each site simultaneously. The ion channel currents were separated temporally or pharmacologically by proper choices of voltage protocols or ion channel inhibitors. Using this strategy we were successful in recording currents from specific ion channel populations. This strategy, which ensured the exact same conditions for the cell lines with respect to Ringer solutions, temperature, pH and osmolarity, allowed a doubling or even tripling of the throughput. We conducted a series of QPatch HTX experiments with a combination of ion channels involved in cardiac risk assessment: hERG, K_v1.5, KvLQT1/minK and Na_v1.5. Specifically, tests were set up for recordings of 1) IV-relationships and concentration-responses for K_v1.5 and Na_v1.5 in parallel by using multiple voltage protocols, and 2) pharmacological properties of specific blockers of hERG, KvLQT1/mink and Nav1.5 in parallel. We present biophysical and pharmacological data obtained with QPatch HTX using multiple voltage protocols and cell lines in combination, and compare them to traditional single-hole data obtained with QPatch HT.

The QPatch HTX automated patch clamp technology was developed to 1) increase throughput in ion channel drug screening by parallel operation of 48 multi-hole patch clamp sites, each comprising 10 individual patch clamp holes, in a single measurements site on a QPlate X, and 2) diminish problems with low-expressing cell lines. Thus, parallel recording from 10 cells represents a 10-fold signal amplification, and it increases the success rate at each site substantially. To further increase throughput we explored the possibility of simultaneous recording of a number of ion channel currents. Two or three cell lines, each expressing a specific ion channel, were applied at each site simultaneously. The ion channel currents were separated temporally or pharmacologically by proper choices of voltage protocols or ion channel inhibitors. Using this strategy we were successful in recording currents from specific ion channel populations. This strategy, which ensured the exact same conditions for the cell lines with respect to Ringer solutions, temperature, pH and osmolarity, allowed a doubling or even tripling of the throughput. We conducted a series of QPatch HTX experiments with a combination of ion channels involved in cardiac risk assessment: hERG, Kv1.5, KvLQT1/minK and Nav1.5. Specifically, tests were set up for recordings of 1) IV-relationships and concentration-responses for Kv1.5 and Nav1.5 in parallel by using multiple voltage protocols, and 2) pharmacological properties of specific blockers of hERG, KvLQT1/mink and Nav1.5 in parallel. We present biophysical and pharmacological data obtained with QPatch HTX using multiple voltage protocols and cell lines in combination, and compare them to traditional single-hole data obtained with QPatch HT.

A large number of mammalian cell lines are commercially available to be used as expression systems for membrane or cytoplasmal proteins. A number of voltage and ligand gated ion channels of potential interest for the pharmaceutical industry are endogenously expressed in several CNS and non-CNS cell lines including TTX-sensitive Na+ channels, Ca2+-release activated Ca2+ (CRAC) channels, inward rectifier K+ channels, acid-sensing ion channels (ASIC) and muscarinic alpha-adrenergic receptors mAChR). We have explored the applicability of five commonly employed cell lines from American Type Culture Collection (ATTC) for use with Sophions QPatchTM automated patch clamp systems (QPatch 16 and QPatch HT) and characterized the ion channel types that they endogenously express. Specificly we have explored:

• Suitability for automated patch clamp studies
  (‘patchability’)
• Background ionic currents that may interfere
  with currents of experimentally expressed ion
  channels
• Possible use for characterizing ion channels of
  interest without the need to experimentally
  introduce their genes (expression)

The tests have led to the development of a number of simple standard operation procedures (SOPs) for employment of the cell lines in QPatch characterizations of ion channels.

Pluripotent stem cell-derived cardiomyocytes, iCells® Cardiomyocytes, were biophysically and pharmacologically characterized with planer automated patch clamp using the QPatch. iCells Cardiomyocytes are differentiated from human induced pluripotent stem cells. They are differentiated in large numbers and cryo-preserved, which make them highly suitable for automated patch clamping and facilitates their use in drug screening. Here we present the results obtained during assay optimization of the cell culture, assay set-up on the QPatch as well as biophysical and pharmacological validation of the cardiac currents. Implementing optimal cell culture routines, the iCells Cardiomyocytes demonstrated spontaneous rhythmical contractions indicating functional properties of adult cardiomyocytes. We tested the cells in two different QPatch recording modes; single-hole recordings and multi-hole recordings where up to ten cells are patched at the same time and the total current is measured per site. Three different types of currents were studied including sodium, calcium and potassium. By using specific buffer solutions and changing the voltage protocols it was possible to characterize calcium, and sodium currents from the same cell. Our study showed that iCells Cardiomyocytes can successfully be applied to the QPatch. The recorded currents are similar to human cardiomyocytes and the response to known pharmacology is as expected. Using the QPatch we believe that iCells Cardiomyocytes have great potential for safety screening and other cardiovascular investigations early in the drug discovery process.

The loss of dopaminergic neurons in the substantia nigra plays an important role in the development of the Parkinson’s disease The symptoms of this disease typically occur after around 80 of these neurons degenerated This cell decay can be caused or promoted by genetic defects or environmental factors
including chemical compounds like pesticides For proper testing of neurotoxic effects on these neurons as well as for the development of neuroprotective drugs, assays based on animal primary cells lack predictivity due to mostly weak correlation between animal and human data. Therefore, models based on
human neuronal cells have a high potential to overcome the limitations of animal models One interesting neuronal cell line is the LUHMES (Lund human mesencephalic) line, which consists of immortalized fetal human mesencephalic cells that can be differentiated into fully postmitotic dopaminergic neurons in 6 days Scholz et al 2011 We here describe functional properties of these cells as a fundament for the
development of LUHMES based pharmacological assays.

hERG (human Ether-a go-go-Related Gene) is a voltage-gated ion channel expressed in cardiac tissue which is of particular concern in off-target pharmacology. Blockade of hERG can lead to prolongation of cardiac repolarisation (long QT syndrome) and ventricular arrhythmia (torsades de pointes). Drug-induced LQTS is the most common reason for drug withdrawal in the last ten years; techniques are therefore required to address this and help to reduce attrition as early as possible in the drug development process.

Second generation automated electrophysiology systems such as IonWorks Barracuda use perforated patch technology to allow c.600 dose-response curves to be generated per day, but produce low fidelity megaOhm recordings with poor success rates. We have developed a more robust, reproducible high throughput whole-cell patch clamp assay methodology using the Sophion Qube 384, which provides higher fidelity
recordings and improved reliability in prediction of activity compared to other automated electrophysiology methods

The ionotropic glutamate receptor GluK1 (previously known as GluR5) is a kainate subtype of the glutamate receptor family and is comprised of a ligand binding domain and an associated ion channel. The receptor is expressed throughout the central nervous system with particularly strong expression in the dorsal root ganglion and trigeminal ganglion; antagonists for the receptor could be treatments for pain disorders, as well as acute migraine.

K2P channels are characterised by their four transmembrane
domain, two-pore topology. Sub-units dimerise to form a
functional channel.

K2P channels carry background (or leak) potassium current in
a variety of cell types and primarily act to maintain resting
membrane potential.

Despite a number of important roles they have proved a
difficult target class to modulate with small molecules.

Thus, there is currently a lack of useful specific
pharmacological tools which target K2Ps. This has limited the
interrogation of the precise physiological function of K2Ps and
efforts to generate K2P targeting therapeutics.

K2Ps have been implicated in a number of human
pathophysiologies including, but not limited to, pain (TREK-1,
TREK-2, TRAAK), migraine (TRESK), Birk-Barel syndrome
(TASK-3) and Balkan endemic nephropathy (TASK-2).

Ligand-gated ion channels are of particular interest to the pharmaceutical industry for the treatment of diseases from a variety of therapeutic areas including CNS disorders, respiratory disease and chronic pain. Ligand-gated ion channels have historically been investigated using fluorescence-based and low throughput patch-clamp techniques. However, the development of the Qube 384 automated patch-clamp system has allowed rapid exchange of liquid and direct measurement of ion channel currents on a millisecond timescale, making it possible to run HTS campaigns and support SAR with a functional readout.

Here, we have used the Qube platform to develop an assay against the NR1/NR2A receptor, which is part of the N-methyl-D-aspartate (NMDA) glutamate receptor family. For this assay, we utilized stacked liquid addition which enabled us to assess the open state kinetics of the channel and to investigate the effects of antagonists with multiple modes of actions.

We observed stable agonist responses for both NMDA and glycine, with EC₅₀ values comparable to literature values. The assay format was stable over multiple agonist applications and wash periods, which meant it was suitable for compound testing and sensitive enough to detect antagonists with multiple modes of action. D-AP-5, a competitive antagonist, showed stable NR1/NR2A receptor over six consecutive agonist/antagonist applications. The potency of ketamine, a use-dependent inhibitor, increased over the six agonist/antagonist applications.

By utilizing of stacked liquid additions in a 384 automated electrophysiology platform we have created an assay against NMDA receptors which is suitable for compound testing and sensitive enough to detect different modes of actions.

Ligand-gated ion channels are of particular interest to the pharmaceutical industry for the treatment of diseases from a variety of therapeutic areas including CNS disorders, respiratory disease and chronic pain. Ligand-gated ion channels have historically been investigated using fluorescence-based and low throughput patch-clamp techniques. However, with the development of the Qube 384 automated patch-clamp system, the rapid exchange of liquid and direct measurement of ion channel currents on a millisecond timescale is now possible at a greater throughput than previously possible. Here, we have used the Qube platform to develop assays against two ligand-gated families: 1) the P2X receptor and 2) the GABAA receptor families. The P2X family is comprised of 7 family members, which are cation permeable and gated by the binding of extracellular ATP. We have assessed both agonist and antagonist pharmacology of 4 members of the P2X family, P2X1, P2X2, P2X3 and P2X4, as well as two species homologs, rP2X3 and gpP2X3. The GABAA α1β3γ2 receptor is a chloride permeable ion channel gated by the binding of GABA. We utilized stacked liquid addition to assess the open state kinetics of the channel and to investigate the effects of a positive allosteric modulator on channel function. As such, we have successfully characterized and developed assays for both the P2X receptor and GABAA receptor families and present EC/IC50 data for antagonists and positive allosteric modulators.

When determining the potencies of drugs in single-cell assays it is a major concern that the employed assay is affected by factors such as the density of the applied cell suspension. It is envisaged that a high density of cells may lead to titration of test compound molecules (by receptor-binding and adsorption) by cells in the vicinity of the targeted cell. This would cause a rightward shift in the concentration-response relationship, and consequently, incorrectly increased IC₅₀ values. We have tested whether IC₅₀ values determined with the QPatch 16 automated patch-clamp system are dependent on the density of the applied cell suspension. We assume that cell density determines the number of cells present in the extracellular flow channel.

It is predicted that increasing the cell suspension density may reduce the time it takes to position a cell on the patch-clamp site. The cell positioning step is accomplished by suction. We tested the effect of the cell suspension density using a wide range of densities (from 0.5 to 16 million cells per ml) on the cell positioning time. For the tests we used two expression systems: (1) CHO cells expressing hERG potassium channels, and (2) HEK cells expressing Na_v1.2a sodium channels. We used test compounds with very different oilwater partition coefficient (log(P)): astemizole and verapamil with log(P) values of 6.43 and 3.45, respectively, (source: www.syrres.com/esc/physdemo) for hERG assays, and tetrodotoxin (TTX) with log(P) of -6.21 for the Na_v1.2a assay.

Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of K_v2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks K_v2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX–fluorophore conjugates colocalize with K_v2.1 clusters in live cells and are released from channels activated by voltage stimuli. K_v2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing K_v2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC–CM) are providing new, highly predictive strategies to assess cardiotoxicity in vitro and can thus reduce costs for cardiac safety assessment in drug development¹.
Different technologies are available to assess compound effects on cardiomyocytes, out of all, the patch clamp technique remains the gold standard as it allows to study compound effects both on individual currents but also on the entire ion channel ensemble in form of an action potential (AP). However, such studies have traditionally been limited by the labour-intensive and low-throughput nature of patch clamp electrophysiology².

Here we present the electrophysiological characterization of hiPSC–CM (Cor.4U) cells using our automated patch clamp (APC) platform Qube 384. Electrophysiological investigation of hiPSC-CM requires a high quality set up that offers the possibility to both record APs in current clamp mode and to isolate individual ion channel currents using voltage clamp. Qube is a 384-channel automated patch clamp system that fulfils all these requirements.

Our data illustrate that stem cell technology in combination with Qube’s high throughput capability holds great potential to accelerate cardiac safety studies.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC – CM) are providing new, highly predictive strategies to assess cardiotoxicity in vitro and can thus reduce costs for cardiac safety assessment in drug development (Ma et al., 2011). In the present study, we characterized Cor.4U® cells using Sophion’s 48-channel QPatch both in voltage and in current clamp mode.

Electrophysiological investigation of hiPSC-CM requires a high quality set up that offers the possibility to both record action potentials in current clamp mode and to isolate individual ion channel currents using voltage clamp.

The QPatch fulfills these requirements and does not rely on the use of a seal enhancer. Many automated patch clamp systems rely on the use of fluoride in the internal solution to gain sufficiently high seal resistances. However, fluoride is also known to chelate Ca²⁺, inhibit proteases and hyperpolarize the gating of sodium channels and may thus affect the recordings (Cummins et al., 2009).

As the number of cardiomyocytes are usually limited, it is critical to minimize the cell consumption per experiment. QPatch is endowed with a feature that helps to reduce the number of cells needed per experiment. Using this feature, 200 μl of cell suspension is sufficient to run a 48-well QPlate.

Human induced pluripotent stem cells (hiPSCs) can be differentiated into multiple cell types, including neurons and cardiomyocytes, which can be used as human disease models for in vitro drug development1. Ion channels represent highly attractive therapeutic targets in the nervous and the cardiovascular system2, rendering electrophysiological studies of hiPSCs important for their usage in drug discovery. However, such studies have traditionally been limited by the labor-intensive and low-throughput nature of patch clamp electrophysiology2.

Here we present the electrophysiological characterization of hiPSC-derived motor neurons using our automated patch clamp (APC) platforms, Qube 384 and QPatch. Our observations include channel expression versus time in culture, the pharmacological dissection of endogenous ion channels (e.g. NaV and KV), identification of ligand-gated receptors and recordings of action potentials.

Utilizing the high-throughput nature of our systems we tested in parallel two disease models, using hiPSC neurons derived from Spinal Muscular Atrophy (SMA) or Amyotrophic Lateral Sclerosis (ALS) patients, together with control cells from healthy subjects and isogenic control cells. The results indicate an over-expression of Na⁺ channels in both cell lines, which could be rescued by a point mutation in the superoxide dismutase (SOD1) gene in the isogenic ALS cell line.

Human-induced pluripotent stem cells (hiPSCs) can be differentiated into multiple cell types, including neurons and cardiomyocytes. This gives rise to a novel way of establishing human disease models, which in turn can be used for drug development in vitro. Ion channels represent highly attractive therapeutic targets in the nervous and the cardiovascular system, rendering electrophysiological studies of hiPSCs important for their usage in drug discovery. However, such studies have traditionally been limited by the labour-intensive and low-throughput nature of patch-clamp electrophysiology. Here we use our automated patch clamp systems Qube 384 and QPatch 48 in order to increase throughput and reduce timelines.

Our observations include channel expression versus time in culture, the pharmacological dissection of endogenous ion channels (e.g. Na_v and K_v), identification of ligand-gated receptors, and recordings of action potentials using the current clamp feature. Also, we show the electrophysiology of a spinal muscular atrophy (SMA) and an amyotrophic lateral sclerosis (ALS) model. The disease model for SMA was derived by mutations in the SMN1 gene and shows enhanced sodium channel activity but no shift in the normalized current-voltage relationship. ALS was here mimicked by a single point mutation in the superoxide dismutase 1 protein (SOD1), D90A, which had previously been identified in recessive, dominant and seemingly sporadic pedigrees. Cells carrying this point mutation displayed larger sodium currents, which eventually led to neurofilament aggregation, neurite degeneration and other phenotypes. We could confirm that the electrophysiological effect could be reversed by point mutation to D90D.

Our measurements validate the feasibility of measuring hiPSC ion channel currents using the APC platforms Qube and QPatch. Altogether, these results can facilitate evaluating the use of hiPSC for early drug development and in extension personal medicine.

The QPatch™ technology has been developed to significantly increase throughput in ion channel drug screening. It is based on planar glass-coated silicon chips with micro-etched patch-clamp holes. Extra andintracellular Ringer solutions are applied by miniature flow channels, which ensures laminar flow and short fluid exchange times (<100 ms). The walls of the flow channels are covered with non-polymer materials (glass and silicon) to minimize problems with non-specific binding of ‘sticky’ compounds. Below is shown the QPatch-16, the first instrument based on the QPatch technology. It runs 16 parallel whole-cell patch-clamp experiments simultaneously with success rates of 50-80 %. Cells are maintained in growth medium in an onboard cell storage facility for up to 4 hours until shortly before an experiment. At that time they are automatically spun down in a miniature centrifuge, washed, and resuspended in Ringer’s solution before being applied to the patch-clamp site. We have determined the IC50 values for 32 hERG channel blockers in a blind test and made a comparison with IC50 values obtained in conventional
manual patch-clamp.

Voltage-gated sodium channels (VGSC) are in the spotlight of drug development as strong evidence is available linking different subtypes to various disease states.

VGSC is responsible for the initiation and propagation of action potentials in excitable cells. During this process, the VGSC transitions from a closed to an open into an inactivated state. Interestingly, inhibitory compounds often exhibit different pharmacological profiles dependent upon the conformational state of the ion channel.

In the present work, the second generation QPatch (QPatch II; Sophion Bioscience) was used in combination with adaptive voltage protocols to investigate state-dependent inhibition of tetrodotoxin (TTX), amitriptyline and tetracaine on 8 different VGSC subtypes (NaV1.1-8). A first step was to determine the half-inactivation potential V½(inactivation) for each individual cell. This value was then used during the next steps as preconditioning pulse. Such an adaptive protocol allowed to determine IC50 values for both the closed and the inactivated state and reduce heterogeneity of the cells. Both IC50 values and biophysical parameters of the different subtypes align well with literature values.

Voltage-gated sodium channels (VGSC) are in the spotlight of drug development as strong evidence is available linking different subtypes to various disease states.

VGSC is responsible for the initiation and propagation of action potentials in excitable cells. During this process, the VGSC transitions from a closed to an open into an inactivated state. Interestingly, inhibitory compounds often exhibit different pharmacological profiles dependent upon the conformational state of the ion channel.

In the present work, the second generation QPatch (QPatch II; Sophion Bioscience) was used in combination with adaptive voltage protocols to investigate state-dependent inhibition of tetrodotoxin (TTX), amitriptyline and tetracaine on 8 different VGSC subtypes (NaV1.1-8). A first step was to determine the half-inactivation potential V½(inactivation) for each individual cell. This value was then used during the next steps as preconditioning pulse. Such an adaptive protocol allowed to determine IC50 values for both the closed and the inactivated state and reduce the heterogeneity of the cells. Both IC50 values and biophysical parameters of the different subtypes align well with literature values.

The new clone screening feature developed for QPatch HT and QPatch 16 allows running up to eight different cell lines (clones or subtypes) at the same time, thus ensuring that the exact same conditions – such as temperature, Ringer’s, pH etc. – are applied for each of the cell lines tested. On QPatch HT each of the eight cell lines are applied to six separate sites on the measurement plate, QPlate 48. After the experiment is finished the data for each cell type can be tracked and compared easily using the advanced analysis functionalities in the QPatch Assay Software. In our study, seven subtypes of the voltage gated sodium channel, Na_v1.1 αβ1, 1.2 α, 1.3 αβ1, 1.4 αβ1, 1.6 αβ1, 1.7 αβ1 and Na_v1.8 α, were tested in parallel on QPatch HT, using the cell cone screening feature. Experiments were designed to explore 1) TTX sensitivity, 2) IV-relationship for activation and inactivation, for the entire panel of Na_v channel subtypes in a single experiment. Thus several different voltage protocols were used in the same experiment.

The voltage dependent sodium channel is responsible for the upstroke and directed propagation of action potentials in nerve and muscle cells and is therefore a central ion channel in excitable tissues. The implication of voltage gated sodium channels in pain mediation and diseases such as epilepsy and cardiac arrhythmia has made them very important targets for drug discovery. Here we show eight subtypes of the voltage gated sodium channel tested in parallel on Qube, the Sophion 384-format automated patch clamp system designed for high throughput and high fidelity electrophysiological recordings. On Qube it is possible to run up to 16 different clones or cell lines simultaneously which ensures identical conditions across the experiments. Three types of experiments were designed to explore 1) TTX sensitivity, 2) IV-relationship for activation and inactivation and 3) pulse train suitable for screening for use dependent sodium channels blockers. For the screening example pharmacology was represented by tetracaine. For every run Nav channel subtypes Nav1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 and 1.8 were tested side by side.

The multi-hole patch clamp technology for the QPatch enables gigaseal recordings of up to ten cells patch-clamped on a single measurement site. In this set of experiments, we have convincingly demonstrated that multi-hole QPatch experiments of fast and slow desensitizing ligand-gated ion channels perform as well as single-hole QPatch experiments with respect to both biophysical and pharmacological characteristics. In the QPlate X, the ten patch holes have a relatively wide spatial distribution to avoid intercellular contact and downstream space clamp issues. The wide spatial distribution could, on the other hand, potentially slow down the liquid exchange times. We examined the glutamate receptor GluR5, the nicotinic acetylcholine receptor nAChR α1, the acid-sensing ion channel ASIC1a, and the anionic γ-aminobutyric acid receptor A GABA-A α1β2γ2 with regards to agonist rise-time, reversal potential and pharmacological properties on the QPatch HTX in multi-hole mode and compared to results obtained in the classic single-hole mode. All data clearly demonstrate that while the amplitude of the elicited ion channel current is multiplied by a factor of 7-10, and the successrate in terms of usable current amplitude is increased, other significant biophysical properties of these ion channels remain unaltered.

The QPatch™ is a powerful ion channel screening system, which was developed through extensive automation of the conventional patch clamp technique. Whole-cell current measurements take place on a disposable 16-channel QPlate™ in MTP format. Later, also a 48-channel QPlate will be available. Automation has been achieved by (i) employing planar silicon chips rather than traditional glass micropipettes, by (ii) elimination and/or simplification of a number of time-consuming tasks, e.g.visual cell selection and pipette tip localisation, manual microscope-aided pipette positioning and cell contact establishment, and subsequent gigaseal and whole-cell formation, and by (iii) cell preparation directly on theQPatch platform. We here report whole-cell currentdata from cultured cell lines expressing a number of voltage- or ligand-gated ion channels. The data were obtained on either complete 16-channel QPlates oron single chip assemblies (1-channel prototype).

The human sodium channel hNa_V1.5, encoded by the SCN5A gene, is crucial for the cardiac action potential upstroke and subsequent signal propagation in the heart. Inhibition of the sodium current by drugs decreases the rate of cardiomyocyte depolarization and the conduction velocity which may lead to serious implications. For these reasons, off-target effects on hNa_V1.5 are considered a risk marker for drug candidates and the hNa_V1.5 is one of the most used in vitro assays in cardiac safety testing and is also one of the most important currents in the CiPA paradigm.

Several drugs, known to inhibit the hNa_V1.5 channel, are either use- or state-dependent and preferentially bind to the open and inactivated state, respectively. When experimenting on voltage-gated ion channels, it is imperative that the voltage applied to the cells is accurate. This is especially important when testing state-dependent compounds. In order to determine compound activity in the most accurate way, all tested cells should have an identical degree of inactivation, a state that will not be achieved by using the same voltage to all experiment sites but rather individual voltages adjusted to the cell’s individual biophysical properties need to be applied.
The Qube, as well as the new QPatch II, are high-throughput automated patch clamp platforms, suitable for studying a wide range of ion channels. Both systems are equipped with the option to run online adaptive protocols, which makes it possible to set each individual cell to a user-defined level of inactivation, e.g. the half-inactivation potential (V_½) and use that value subsequently in e.g. a preconditioning pulse. Applying this new adaptive protocol feature, we determined Na_V1.5 IC₅₀ values for both the open and the inactivated state for a set of known sodium channel inhibitors. We could show that the use of individual V_½ reduces data variability compared to standard methods and thereby improves the accuracy of drug evaluation.

The suitability of an automated patch clamp for the characterization and pharmacological screening of calcium release–activated calcium (CRAC) channels endogenously expressed in RBL-2H3 cells was explored with the QPatch system. CRAC currents (ICRAC) are small, and thus precise recordings require high signal-to-noise ratios obtained by high seal resistances. Automated whole-cell establishment resulted in membrane resistances of 1728 ± 226 MΩ (n = 44). CRAC channels were activated by a number of methods that raise intracellular calcium concentration, including EGTA, ionomycin, Ins(1,4,5)P3, and thapsigargin. ICRAC whole-cell currents ranged from 30 to 120 pA with rise times of 40 to 150 s. An initial delay in current activation was observed in particular when ICRAC was activated by passive store depletion using EGTA. Apparent rundown of ICRAC was commonly observed, and the current could be reactivated by subsequent addition of thapsigargin. ICRAC was blocked by SKF-96365 and 2-APB with IC₅₀ values of 4.7 ± 1.1 μM (n = 9) and 7.5 ± 0.7 (n = 9) μM, respectively. The potencies of these blockers were similar to values reported for ICRAC in similar conventional patch-clamp experiments. The study demonstrates that CRAC channels can be rapidly and efficiently targeted with automated patch-clamp techniques for characterization of physiological and pharmacological properties.

In cardiac muscle, sustained inward Na+ currents, also known as late Na⁺ currents (I_Na-L), occur under physiological conditions during the cardiac Action Potential AP (AP). In addition to extending the plateau duration before AP recovery, an increased INa-L can lead to the development of various triggers and substrates (early after depolarization, EAD) for arrhythmogenesis. Inhibition of I_Na-L current can prevent proarrythmia by reducing or reversing EAD even in the presence of a prolonged QTc interval (e.g. Ranolazine).

Therefore, I_Na-L is one of the seven ion channels selected for
evaluation in the comprehensive in vitro proarrythmia assay (CiPA).

Eurofins is a contributor to the Ion-Channel Work-Group of the
FDA/HESI/CIPA initiative, supporting validation of automated high-throughput methods on the QPatch HT to support the program consortium. These methods measure 1) enhancement or 2) inhibition of I_Na-L current, using either step pulse or ramp pulse protocol and reference pharmacology, ATXII. Three key parameters, I_Na-L current, leak current, time-matched vehicle control were measured. The data generated in this study demonstrate that measuring I_Na-L current charge measurement is superior to measuring I_Na-L current amplitude for both test and ramp pulse. We investigated the effects of CiPA-28 compounds on I_Na-L at the EC₅₀ of ATXII (30nM). With the un-blinding of CiPA Phase I (12) compounds, Chlorpromazine, Diltiazem, and Mexiletine were identified as hits. Our results indicate that none of the CiPA-28 compounds potentiated the I_Na-L current.

Most ligand-gated ion channels (LGICs) are characterized by fast transient currents in response to application of agonists. Typically the time constants for activation and subsequent desensitization amount to1-100 and 10-1000 ms, respectively. Consequently, recording of proper whole-cell LGIC currents with the patch clamp technique requires a fast solution exchange system. Furthermore, characterization of LGIC blockers and modulators generally requires complex compound application protocols, because the effect of a test compound needs to be evaluated simultaneously with a tran- sient application of the agonist. These requirements challenge a realistic electrophysiological characterization of LGICs. We employed the automated QPatch 16 patch-clamp system, to characterize the effects of agonists, antagonists and activators on two types of fast LGICs: (1) GABAA (γ- amino-butyric acid A) receptors and (2) ASIC (acid sensing ion channels) types 1a and 3.

ICH guidelines state that compounds in drug discovery must be tested for inhibition of hERG cardiac
ion channel. It is often prudent to test compounds against a wider array of cardiac ion channels, e.g.
hNa_V1.5 and hCa_V1.2 (Kramer et al., 2013). The CiPA initiative will demand testing and additional ion
channel targets as well; namely hNa_V1.5 late current, hKir2.1, hKvLQT1 and Kv4.3 (Gintant et al., 2016).
These ion channel assays are all amenable to automated patch-clamp and have typically been run
using recombinant cell lines over-expressing an individual ion channel. The aim of this research was to
investigate whether human induced pluripotent stem cell-derived cardiomyocytes are a useful,
affordable and predictive cellular reagent for use on the QPatch automated patch-clamp system.

The QPatch 16 screening station is a second generation automated patch-clamp system based on planar silicon chip technology. QPatch 16 has previously been employed for a series of screening studies on a number of voltage-gated ion channels (e.g. hERG, KCNQ4, Na_v1.2, Na_v1.4 and Na_v1.5). Recently, ligand-gated ion channels (LGIC) including GABAA, nAChR and ASIC have been targeted with QPatch 16. We here report a study in which the GABAA receptor (α1 β2 γ2) was targeted with 4-5 concentrations of an agonist (GABA), an antagonist (bicuculline) and a modulator (chlordizepoxide). Subsequently we report a study on acid sensitive ion channels (ASIC1a) in which the effect of pH, i.e. protons which serve as the ligand, was examined.

Voltage-gated sodium (Na_V) channels are studied extensively due to their potential as targets for several indications, such as pain, epilepsy, cardiac and muscle paralysis. Some Na_V channel modulators show state-dependence and bind preferentially to the inactivated state of the channel. The potency of state-dependent compounds is known to vary depending on the percentage inactivation of the channels.

To calculate accurate compound activity the precise value for the VHalf of inactivation should be used for each cell. The adaptive protocol block for the Sophion Qube 384-well automated patch clamp platform has made it possible to separately define the voltage applied to individual wells for both the activation and inactivation of the channels. This enables the generation of more precise data for voltage-gated ion channels.

Revising the ICH regulatory guideline, S7B, for the Non-clinical Evaluation of the Potential for Delayed Ventricular repolarization (QT Interval Prolongation by Human Pharmaceuticals) has been suggested and Comprehensive In Vitro Proarrhythmia Assay (CiPA) activities are being implemented. We previously studied the applicability of the CiPA-recommended voltage protocols for hERG, Cav1.2, and late Nav1.5 current patch clamping which was announced last year (this research was presented at the 2019 Japanese Safety Pharmacology Society annual meeting). As a result, inter-facility differences were found for some compounds in the hERG assay on an automated patch-clamp system, QPatch. Such differences were also noted in enhancement ability of ATX-II for late Nav1.5 current. This time, we investigated the factors for the interfacility differences and studied more appropriate protocols. In addition, we present the results of the assays by the CiPA-recommended protocols for each ion channel. Manual patch clamp and QPatch system were selected as the platforms. For the voltage protocols, the CiPA-recommended ones were followed whereas the composition of intra- and extracellular buffers were modified. Preparation procedures and application methods of test solutions (such as application duration or number of replicates) for the QPatch assay were examined. Test compounds were selected from each of the low-, intermediate-, and high-risk categories of the “training drugs” in the CiPA in silico model. We present the comparison results of our data by the CiPA-recommended protocols and the data given by the CiPA. We also discuss buffer compositions, preparation procedures or application methods for test solutions to reduce the inter-facility differences. We would like to show and discuss the more appropriate protocols including the detailed experimental methods, which are not given in the CiPA-recommended protocols, and points to be modified.

Kv4.3 α-subunits associate with ancillary β-subunits of the KChIP2 family in heart muscular tissue to channel transient outward (Ito) currents. Decreased expression or dysfunction of Kv4.3 channels following myocardial infarction or during heart failure can contribute to abnormal repolarization, which may result in ventricular arrhythmias. Therefore, drug-induced inhibition of Kv4.3/KChIP2-mediated Ito exposes to potential cardio safety liabilities which are important to document early during the drug discovery process. In this study, we have characterized basic electrophysiological properties pertaining to Ito currents in CHO cells expressing recombinant Kv4.3 and KChIP2.2 subunits. The currents obtained were validated pharmacologically by assessing the efficacy of small molecule and peptide toxin inhibitors known from manual patch-clamp studies to block Ito. We then examined the inhibitory activities of 28 drugs with clinically documented high, medium or low pro-arrhythmic risk encompassing the test- and validation-sets of the Comprehensive in vitro Pro-Arrhythmia (CiPA) panel. While none of the CiPA drugs inhibited Kv4.3 at submicromolar concentrations (IC50 < 1 µM), two drugs (loratadine and nitrendipine) displayed intermediate potency (1 µM < IC50 < 10 µM), and four drugs (bepridil, quinidine, chlorpromazine and astemizole) revealed weak, but significant (10 µM < IC50 < 30 µM) inhibitors.

Sodium ion channels are some of the fastest ion channels. With a high current and fast activation, it is very difficult for conventional amplifiers to correctly clamp these signals. The main challenge in providing a perfect clamp is the error introduced by the series resistance (Rs), which will introduce a voltage drop as the current increases rapidly (Sigworth 1983). This leads to delays in the signal and incorrect estimation of the current magnitude. In order to correctly compensate Rs fast enough, one will have to compromise on another cell parameter; cell capacitance (Cs) (Sherman 1999). In the QPatch system, it is possible to conduct experiments without direct Cs compensation, but still measure Cs for later use.

When compensating completely for Rs, oscillations may occur and this is mainly due to incorrect cell parameter estimations or that the cell simply changes slightly. Oscillations can often lead to reduction in the seal resistance and terminate the whole-cell configuration. In the QPatch system, critical current oscillations are detected and the Rs feedback loop is disconnected for the remaining of the sweep until new parameter estimations can be made.