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Drug discovery is challenged by poor predictability of proarrhythmic effects of present safety screening approaches. The Comprehensive in Vitro Proarrhythmia Assay (CiPA), an FDA-directed initiative, is specifically focused on improved preclinical assessment of torsade de pointes (TdP) risk, striving to optimize the drug discovery process.In recent years, human stem cell-derived cardiomyocytes have proven to recapitulate key features of human cardiac electrophysiology in vitro. Furthermore, it has become apparent that the intact ensemble of cardiac ion channels is necessary to determine proarrhythmic effects reliably. Hence, due to their relatively easy availability, stem cell-derived cardiomyocytes have become the preferred choice of cardiac cells.We present chip-based approaches allowing parallel patch clamp recordings without compromising data quality or technical sophistication. Culture and harvesting protocols have been optimized on cardiomyocytes for success rate but also the number of cells used.We show patch clamp data from two automated patch clamp platforms (medium throughput - Patchliner and high throughput - SyncroPatch 384/768PE). The Patchliner and the SyncroPatch 384/768PE both boast temperature control to enable recordings at physiological temperature - an important feature since drug efficacies may vary with temperature. Here, we present pharmacological data on voltage-dependent channels of the CiPA panel at different temperatures.The effect of drugs on action potentials is important for assessing the interaction of the cardiac ion channel ensemble. Both patch clamp platforms have amplifiers capable of current clamp. Compound effects on action potentials will also be presented.Patchliner and SyncroPatch 384/768PE are both planar patch-clamp platforms combining all the necessary features for successful drug discovery (well-suited for stem cell-derived cardiomyocytes, temperature control, and current clamp mode).

The inhibitory effects of the anesthetic barbiturate pentobarbital on the slow (I Ks) and fast component (I Kr) of cardiac delayed rectifier potassium currents (I K) and on the inward rectifier potassium currents (I K1) were examined in Xenopus oocytes expressing the human minK, human ether-a-go-go related gene (HERG) and guinea pig Kir2.2, respectively. Block of native I K (I Ks and I Kr) and inward rectifier potassium current (I K1) by pentobarbital was examined in guinea pig ventricular myocytes. In oocytes using the two electrode voltage clamp technique potassium currents of hminK-, HERG- and Kir2.2-expressing oocytes were inhibited by pentobarbital with IC50 values of 0.20, 1.58 and 0.54 mM, respectively. I Ks block was time- and voltage-independent and had no influence on activation at positive voltages although it shifted voltage-dependent activation to more positive voltages. Pentobarbital-induced HERG inhibition was not dependent on voltage but influenced the deactivation kinetics and shifted half-maximal activation to more negative voltages. In guinea pig cardiomyocytes, using the patch clamp technique, I Ks and I Kr were inhibited by pentobarbital with IC50 values of 0.18 mM and 2.75 mM, respectively. I Kr deactivation and I Ks activation kinetics were only slightly influenced by pentobarbital, if at all. Block of I K1 was weakly voltage-dependent with IC50 values of 0.26 mM (–40 mV) and 0.91 mM (–120 mV). The data show that pentobarbital suppresses both cloned (I K, I Kir2.2) and native (I K, I K1) cardiac potassium currents with the highest affinity for I Ks.

Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (NaV1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of NaV1.7, and activation and allosteric modulation of nAChα7R. Curr. Protoc. Pharmacol. 65:11.13.1-11.13.48. © 2014 by John Wiley & Sons, Inc. Keywords: automated patch clamp; NaV1.7; nicotinic acetylcholine receptors

The temperature-sensitive ion channel family is receiving increasing attention as a potential contributor to pain states. In particular, ion channels activated by noxious heat, for example the TRPV1 channel, could provide a novel target for the treatment of chronic pain.Using an automated patch clamp system, TRPV1 receptors expressed in CHO cells were activated using either noxious heat or the ligand capsaicin. Antagonists can be used to block either the heat- or capsaicin-activated TRPV1 response. Since blocking the heat response of TRPV1 can have undesirable effects on core body temperature in animals and humans, being able to discriminate between antagonising the ligand-activated vs. the heat-activated response of TRPV1 channels may be important for discovering novel compounds with a reduced side-effect profile. Data will be shown of TRPV1 currents activated by increasing temperature and block of the heat-activated response by ruthenium red. Data will also be presented showing different antagonist profiles for ligand- and heat-activation of TRPV1.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Since the discovery of the possible solubilization of membrane proteins and their isolation from other membrane constituents (purification), different methods were developed to reconstitute ion channel proteins into artificial lipid bilayers. These membrane proteins were then fully functional when correctly oriented and inserted in a lipid bilayer. The reconstitution plays a central role in identifying and characterizing the mechanisms of action of membrane proteins. The activity of the membrane proteins is studied using electrophysiology using different methods e.g., the black lipid membrane. Therefore, the structure-function relationship can be investigated to better understand the biophysical properties of membrane proteins in vivo.Using a glass surface containing a micrometer hole, the fusion of vesicles on the surface becomes an attractive method for electrophysiology and then to reconstitute membrane proteins into the lipid bilayer without denaturation. Then stable lipid bilayers are formed by bursting a GUV on the glass surface, forming a free-standing portion above the hole.With this technique, we studied the biophysical and pharmacological properties of different ion channels, for example potassium channels (KcsA, Kv1.2), sodium channels (NachBac, NaVsp1) as well as other ligand-dependent (IP3 receptor, NMDA receptor), mechanosensitive channels (MscL, TRP channels) and non-specific channels (Cx43, VDAC). I will describe here our methods for incorporation of proteins into the bilayer and the recording of single ion channel current measurements by using a planar patch clamp platform.