Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na(+) (Na(v)), Ca(2+) (Ca(v)), and K(+) (K(v)) channels as expected from excitable cells. The Na(v) current was TTX sensitive, IC(50) = 12 ± 6 nM (n = 5). About 50% of the Ca(v) current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the K(v) channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC(50) = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABA(A) receptor antagonist bicuculline, IC(50) = 632 ± 149 nM (n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed Na(v) currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching "excitable cells." These results show high potential for pluripotent stem cell-derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.

IntroductIon Studying cardiac action potential modulation is important for preclinical drug safety testing. it offers a rationale for potentially required medicinal chemistry efforts, lead compound optimization, or project termination due to cardiotoxic side effects.the patch clamp technique is the gold standard for real-time investigation of ion channels, 1 and the automation of the method increases the throughput and makes it accessible to a wider audience due to its ease of use compared with the conventional patch clamp method. 2 to overcome the hurdles in working with primary heart tissue from human donors or animals, the use of stem cell-derived cardiomyocytes may be a viable option for safety screening of larger-compound libraries in the earlier stages of the drug development process.Here we describe the electrophysiological characterization of mouse embryonic stem cell (meSc)-derived cardiomyocytes on an automated patch clamp platform with voltage clamp and current clamp capability for assessing action potentials and their pharmacological modulation.these cardiomyocytes are readily available and 100% pure, in contrast to preparations of primary cardiac myocytes, which are usually contaminated by fibroblasts. 3 moreover, the selected meSc-derived cardiomyocytes revealed their physiological relevance by functional integration into in farcted mouse hearts and prolonging the span of life of transplanted mice in comparison with the control group. 4 We show that these meSc-derived cardiomyocytes functionally express all essential cardiac ion channels and that typical cardiac action potentials can be elicited.the combination of an automated patch clamp instrument together with a standardized and pure cardiac myocyte model enables scientists in basic or applied cardiology and toxicology to perform a cost-and time-effective screening. cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. to identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. the aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. the authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automa...

Background: Species-dependent pharmacology is an obstacle for TRPV1 antagonist development. Results: By exchanging the pore domains TRPV1 antagonist JYL-1421, which is modality-selective in rTRPV1 can be made modality-selective in hTRPV1 and vice-versa. Conclusion: The pore region is critical for the observed species differences. Significance: Thus, the findings are of significance for the development of more specific and selective TRPV1 antagonists.

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately six years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world's smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion's higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and Nav1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

The hERG gene encodes a potassium channel responsible for the repolarisation of the IKr current in cardiac cells. Given the importance of this channel in the repolarisation of the cardiac action potential, and the disturbances of channel function by certain compounds such as anti-arrhythmias and anti-psychotics, this channel has become very important in safety pharmacology testing. Since some hERG-active compounds also exhibit different pharmacology at physiological temperature, experiments performed at this temperature are important in yielding more relevant data in safety screening. In this study, we describe the use of automated patch clamp electrophysiology for recording hERG stably transfected in HEK293 cells. Recordings of the hERG current from up to eight cells simultaneously could be performed at room temperature (RT) and at physiological temperature. Data will be shown for erythromycin which exhibited a higher potency at 35°C vs RT. Additionally, using a planar patch clamp workstation, recordings could be made from stem cell-derived cardiomyocytes. Currents mediated by K+, Na+ and Ca2+ channels could be recorded in the voltage clamp mode. What is more, action potentials in the current clamp mode could also be recorded and pharmacology was performed on action potentials (data will be shown). The use of stem cell-derived cardiomyocytes in safety testing is becoming increasingly important. The ability to test compounds on ion channels in both the voltage and the current clamp modes, as well as at different temperatures, may be crucial for future safety testing.