Voltage- and current clamp on induced pluripotent cardiomyocytes with automated patch clamp
Biophysics Go to journalAuthor(s):
Daniel Sauter, Anders Lindqvist, Rasmus Bjørn Jacobsen, Jens Henneke, Mads P.G. Korsgaard
Use- and state-dependent Nav1.5 blockers on QPatch X and in vivo and in vitro assays
SPS 2010 Go to journalAuthor(s):
M Knirke Jensen, Tomoko Sakakura, Yasuyuki Abe, Hideo Takamori, Kiyoshi Takasuna, Yuji Tsurubuchi, Morten Sunesen
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 Nav1.5. 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 INa (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 INa (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 INa (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 Nav1.5 blocking activity and no proarrhythmic activity, and 2) the pro-arrhythmic activity of the Nav1.5 blockers flecainide and quinidine might be attributable to their marked delay of recovery from inactivation.
Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery
Receptors Channels Go to journalAuthor(s):
M Asmild, N Oswald, KM Krzywkowski, S Friis, RB Jacobsen, D Reuter, R Taboryski, J Kutchinsky, RK Vestergaard, RL Schrøder, CB Sørensen, M Bech, MP Korsgaard, NJ Willumsen
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.
TRP’ing in multi-hole mode
Biophysics 2010 Go to journalAuthor(s):
Rasmus B Jacobsen, Hervør L Olsen
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
Transient expression of the NMDA receptor in HEK cells co-expressing the glutamate transporter for the screening of potential positive allosteric modulators of NR2B
Drug Discovery Go to journalAuthor(s):
Michael J Morton, John Malone, Paul Sharpe, Graham Sproat, Malcolm Haddrick, Joe Mather
TMEM16A on Qube 384
Ion Channel Modulation Symposium 2017 Go to journalAuthor(s):
Joseph G. McGivern*, John K. Sullivan*, Kathryn A. Henckels*, Paul Wang*, David Powers*, Daniel Sauter**, Lars Løjkner**, Rasmus Jakobsen**, Mads Korsgaard**
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)
Temperature effect on hERG channel pharmacology measured using the Qube automated patch clamp system
Sophion Poster Go to journalAuthor(s):
Anders Lindqvist, Mette Christensen, Rasmus Jacobsen, Göran Mattson
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.
Strategies for enhancing throughput in ion channel drug screening
Biophysics 2008 Go to journalAuthor(s):
Rasmus B. Jacobsen, Naja M. Sørensen, Hervør L. Olsen, M. Sunesen, *Niels J. Willumsen
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:
Application of multiple drugs per cell
Reduction of the duration of the experimental
Estimation of drug potency (IC50) based on a
single inhibitor concentration
Sophion analyzer as a tool for biophysical and pharmacological characterization of eight Nav subtypes evaluated in parallel on Qube
Neuroscience 2016 Go to journalAuthor(s):
Mads P. G. Korsgaard, Lars Damgaard Løjkner, Anders Lindqvist, Mette T Christensen, Thomas Binzer
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.
Simple and Effective Generation of Cell-based Assays for Ion Channels, Transporters, and Kinase Screening in Biologically Relevant Cells Using Scalable Transient Transfection
ChanTest Conference, May Go to journalAuthor(s):
Qin (Ching) Chen, James Brady, Karen Donato, Meg Duskin, and Madhusudan Peshwa
Scaffolds within scaffolds: Generating ion channel blocking antibodies by fusing knottin to peripheral CDR loops
IONTAS Go to journalAuthor(s):
A.Karatt-Vellatt1, S. Surade1, D. C. Bell1, T. Leutkens1, E. Masters1, A.M. Luther1, N.M. Sørensen2, N. Butt1 & J. McCafferty1
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.
Rapid generation of cells for ion channels assays: Efficient, large scale transfection using the MaxCyte STX
MaxCyte Go to journalAuthor(s):
James Brady, Peer Heine, Rama Shivakumar, Angelia Viley, Madhusudan Peshwa, Karen Donato and Krista Steger - MaxCyte
Mark Rothenberg. Corning Inc., Kennebunk, ME, USA. Hervør Lykke Olsen, Nikolaj Nielsen, Kristina Christensen, Jeffrey Webber and Morten Sunesen - Sophion Bioscience
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.
Qube as a tool for assay optimization of CiPA cells and protocols by using multiple IC, EC solutions and hERG, Nav1.5 and Cav1.2 on the same QChip
SPS 2016 Go to journalAuthor(s):
Melanie Schupp, Anders Lindqvist, Pia Bladsgaard Andersen, Weifeng Yu
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 Nav1.5 (peak and late currents), Kv4.3 (Ito), hERG (IKr), KvLQT1/minK (IKs), Cav1.2 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 Nav1.5, Cav1.2, Kv1.5 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.
Qube 384-channel patch clamp characterization of the liquid flow channels
SLAS 2013 Go to journalAuthor(s):
Rasmus B Jacobsen, Søren Friis, Hervør L Olsen, Mette T Christensen, Kristina M Christensen, Peder S Pedersen, Anders Hyldgaard, Morten Sunesen, Anders Lindqvist
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.
Qube 384 channel patch clamp screening system ligand gated ion channel applications
Biophysics 2013 Go to journalAuthor(s):
Søren Friis, Rasmus B Jacobsen, Hervør Lykke Olsen, Mette T Christensen, Kristina M. Christensen, Peder Skafte Pedersen, Lasse Homann, Morten Sunesen, Anders Lindqvist
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.
Preparation, Antiepileptic Activity, and Cardiovascular Safety of Dihydropyrazoles as Brain-Penetrant T-Type Calcium Channel Blockers
Journal of Medicinal Chemistry Go to journalAuthor(s):
Lubos Remen†, Olivier Bezençon*†, Lloyd Simons‡, Rick Gaston‡, Dennis Downing‡, John Gatfield†, Catherine Roch†, Melanie Kessler†, Johannes Mosbacher†, Thomas Pfeifer†, Corinna Grisostomi†, Markus Rey†, Eric A. Ertel†, and Richard Moon†
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.
Pharmacological characterization of voltage- and ligand-gated ion channels
JSPS 2006 Go to journalAuthor(s):
Hervør L Olsen, Rasmus B Jacobsen, Søren Friis, Morten Sunesen, Niels Willumsen, Rikke Schrøder, Naja M Sørensen, M Knirke Jensen
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.
Parallel patch clamp of alpha 7 nicotinic acetylcholine receptor channels
SfN 2007 Go to journalAuthor(s):
Søren Friis, Morten Sunesen, Chris Mathes, Mark Bowlby, John Dunlop, Niels Willumsen
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.
Optimizing CHO hERG for automated patch clamping
Sophion Bioscience Go to journalAuthor(s):
Søren Friis, Hervør L Olsen, Jeffrey Webber, M Knirke Jensen, Dorthe Nielsen, Morten Sunesen, Chris Mathes
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.
High Throughput Electrophysiology: New Perspectives for Ion Channel Drug Discovery
Receptors and Channels Go to journalAuthor(s):
Niels J. Willumsen, Morten Bech, Søren-Peter Olesen, Bo Skaaning Jensen, Mads P. G. Korsgaard, and Palle Christophersen
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.
Exploring stem cell-derived cardiomyocytes with automated patch clamp techniques
Biophysics 2012 Go to journalAuthor(s):
Rikke P Schrøder, Mette T Christensen, Blake Anson, Morten Sunesen
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.
Evaluation of throughput and data quality
Biophysics 2009 Go to journalAuthor(s):
Hervor L Olsen, Rikke P Schrøder, Rasmus B Jacobsen, Søren Friis, Morten Sunesen
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 Nav1.2a. This stable cell line is characterized by a very low functional expression of the Nav1.2a 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 Nav1.2a 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.
Enhancing throughput with multiple cell lines per well with the QPatch HTX
SBS 2010 Go to journalAuthor(s):
Hervor L Olsen, Rikke P Schrøder
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.
Enhancing throughput with multiple cell lines per well
Sophion Bioscience Go to journalAuthor(s):
Hervor L Olsen, Dorthe Nielsen, Morten Sunesen
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.
Endogenous Ion Channels of Mammalian Cell Lines Characterization
Sophion Bioscience Go to journalAuthor(s):
Søren Friis, Rasmus B Jacobsen, Morten Sunesen, Dorthe Nielsen, Nadia Larsen, Chris Mathes, Niels Willumsen
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
Background ionic currents that may interfere
with currents of experimentally expressed ion
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.
Electrophysiological properties of iCell cardiomyocytes obtained by automated patch clamp
SPS 2011 Go to journalAuthor(s):
Rikke P Schrøder 1), Mette T Christensen 1), Giorgia Salvagiotto 2), Blake Anson 2), Morten Sunesen 1)
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.
Electrophysiological characterization of human induced pluripotent stem cell-derived dopaminergic neurons using manual and automated patch clamp systems
Neurorepair Go to journalAuthor(s):
Denise Franz, Jan Giemsa, Oliver Klink, Hervør Lykke Olsen
Development of an Ultra High-Throughput Screen to Identify Antagonists for GluK1
Merck Go to journalAuthor(s):
Kelli Solly1 , Carissa Quinn1 , Rebecca Klein2 , Darrel Henze 2 , Eric Johnson1 , and Michael Finley 1
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.
Data quality is not affected by cell suspension density
Sophion Bioscience Go to journalAuthor(s):
Chris Mathes, Hervør L Olsen, Michael Stoltenborg, Morten Sunesen, Niels Willumsen
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 Nav1.2a 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 Nav1.2a assay.
Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells
PNAS Go to journalAuthor(s):
Drew C. Tilleya (1, Kenneth S. Euma, (b,1,2, Sebastian Fletcher-Taylora, Daniel C. Austina, Christophe Dupréb, Lilian A. Patrónb, Rita L. Garciac, Kit Lamd, Vladimir Yarov-Yarovoya,d, Bruce E. Cohenc, and Jon T. Sacka,b (3
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 Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 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 Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 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 Kv2.1, 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.
Characterization of human iPSC-derived cardiomyocytes (Cor.4U) on an automated planar patch clamp set-up (QPatch)
Sophion poster Go to journalAuthor(s):
Daniel Sauter, Kim Boddum, Kazuya Tsurudome, Rasmus B Jacobsen, Göran Mattsson
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.
Characterization of hERG blockers using the automated QPatch 16 screening system
Biophysics 2005 Go to journalAuthor(s):
Rasmus B Jacobsen, Simon Pedersen, Søren Friis, Nicoline Andersen, Niels Willumsen
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
Biophysical and pharmacological profiling of multiple Nav subtypes on QPatch HT
Biophysics 2016 Go to journalAuthor(s):
Anders Lindqvist, Søren Friis, Mette T. Christensen & Mads P.G. Korsgaard
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, Nav1.1 αβ1, 1.2 α, 1.3 αβ1, 1.4 αβ1, 1.6 αβ1, 1.7 αβ1 and Nav1.8 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 Nav channel subtypes in a single experiment. Thus several different voltage protocols were used in the same experiment.
Biophysical and pharmacological characterization of multiple Nav subtypes on Qube
Biophysics 2016 Go to journalAuthor(s):
Anders Lindqvist, Søren Friis, Mette T. Christensen & Mads P.G. Korsgaard
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.
Biophysical and pharmacological characterization of ligandgated ion channels in multi-hole mode
Neuroscience SfN 2010 Go to journalAuthor(s):
M. Knirke Jensen, Rikke Schrøder, Hervør L. Olsen, Rasmus B. Jacobsen, Jeffrey Webber, Søren Friis, Dorthe Nielsen, Mette T. Christensen, Morten Sunesen
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.
Automation of patch clamp techniques: Technical validation through characterization of voltage-gated potassium channels and ligand-gated ion channels
Biophysics 2004 Go to journalAuthor(s):
Karen Krzywkowski, Rikke Schrøder, Trine Ljungstrøm, Jonatan Kutchinsky, Søren Friis, Margit Asmild, Rafael Taborski, Simon Pedersen, Ras K. Vestergaard, Rasmus B Jacobsen, Nathalie Helix, Claus B Sørensen Morten Bech, Jeppe K Christensen, Simon du Beau, Niels Willumsen
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).
Automated Patch-Clamp Technique: Increased Throughput in Functional Characterization and in Pharmacological Screening of Small-Conductance Ca2+ Release-Activated Ca2+ Channels
Sage Journals Go to journalAuthor(s):
Rikke L. Schrøder 1), Søren Friis 1), Morten Sunesen 1), Chris Mathes 2), and Niels J. Willumsen 3)
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.
Assessing functional properties of ligand-gated ion channels with automated whole-cell patch clamp technology
Biophysics 2006 Go to journalAuthor(s):
Hervør L Olsen, Rasmus B Jacobsen, Rikke Schrøder, Jonatan Kutchinsky, Morten Sunesen, Niels Willumsen
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 require- ments challenge a realistic electrophysiological characterization of LGICs. We employed the auto- mated QPatch 16 patch-clamp system, to charac- terize the effects of agonists, antagonists and acti- vators 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.
Are stem-cell cardiomyocytes a viable cellular reagent for automated patch-clamp?
Axol Bio Go to journalAuthor(s):
Michael Morton, Lauren Eades
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.
hNaV1.5 and hCaV1.2 (Kramer et al., 2013). The CiPA initiative will demand testing and additional ion
channel targets as well; namely hNaV1.5 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.
Application of QPatch 16 for drug screening of ligand-gated ion channels
SBS 2005 Go to journalAuthor(s):
Nicoline R. Andersen, Simon Pedersen, Jeppe K. Christensen, Michael Stoltenborg, Morten Sunesen, Niels J. Willumsen
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, Nav1.2, Nav1.4 and Nav1.5). 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.
“Clip Detecting” with series resistance compensation using an automated patch clamp system
Biophysics 2012 Go to journalAuthor(s):
Lars Damgaard Løjkner, Chris Gibbs, Jesper Gerved, Jens Henneke, Jonatan Kutchinsky, Rasmus B Jacobsen, Morten Sunesen
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.