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Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na+/glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip. In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition. Sugar concentration jumps in the presence of sodium lead to transport currents between 5 and 10 nA. Remarkably, in the absence of sodium (i.e. no transport), we observed fast pre-steady-state (PSS) currents with time constants between 3 and 10 ms. These PSS currents mainly originate from sugar binding. Sodium binding does not induce PSS currents. Due to high time resolution, PSS currents were distinguished from transport and eventually correlated with conformational transitions within the sugar translocation pathway. In addition, we analyzed the impact of driving forces on transport and binding currents, showing that membrane voltage and sodium concentration gradients lead to an increased transport rate without affecting sugar binding kinetics. We also compared Na+/sugar efflux with physiologically relevant influx and found similar transport rates, but lower affinity in efflux mode. SSM-based electrophysiology is a powerful technique, which overcomes bottlenecks for transport measurements observed in other techniques such as the requirement of labels or the lack of real-time data. Rapid solution exchange enables the observation of substrate-induced electrogenic events like conformational transitions, opening novel perspectives for in-depth functional studies of SGLT1 and other transporters.

We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration-response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNaV1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNaV1.7 and hERG is described. Fast external solution exchange rates of 200 MΩ, and recordings of voltage-gated Na+ and Ca2+ are shown.

Introduction Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. Methods APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. Results All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature ( Kramer et al., 2013 ) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2–3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. Discussion Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.

The need for predictive, in vitro cardiac safety screening drives further development of automated, high-throughput-compatible drug evaluation based on cardiac cell preparations. Recently, pluripotent stem cells are evaluated as a new, more predictive model for cardiovascular risk assessment pertaining to in vitro assays. We present a new screening platform, the CardioExcyte 96, a hybrid instrument that combines impedance (cell contractility) with extracellular field potential (EFP) recordings. The electrophysiological measurements are noninvasive, label free and have a temporal resolution of 1 ms. This hybrid technology addresses the lack of easy-to-use high-throughput screening for in vitro assays and permits the reliable investigation of short- and long-term pharmacological effects. Several models of cardiomyocyte preparations were successfully validated for use with the CardioExcyte96. Furthermore, the pharmacological effects of a number of reference compounds were evaluated. Compound effects on cell monolayers of human-induced pluripotent stem cell-derived cardiomyocytes are evaluated using a quasi-simultaneous hybrid recording mode that combines impedance and EFP readouts. A specialized software package for rapid data handling and real-time analysis was developed, which allows for comprehensive investigation of the cellular beat signal. Combining impedance readouts of cell contractility and EFP (microelectrode array-like) recordings, the system opens up new possibilities in the field of in vitro cardiac safety assessment.

The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using 14 C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba 2+ ). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90 min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.

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).

Multi-drug resistant (MDR) bacteria are an increasingly serious threat to global public health. In the last decades, many studies showed that general outer membrane porins (OMPs) from Gram-negative bacteria represent the major route for antibiotics to translocate through the outer membrane.To explore the molecular rules for efficient drug transport and therefore cell killing, molecule-protein interaction is analyzed at single molecule level via electrophysiology. We incorporated OMPs into planar bilayer and single-channel currents were measured by an automated planar lipid bilayer station (Orbit 16, Nanion). The role of OMPs in the molecule-protein interactions can be characterized by the interaction kinetics. Here, we show by using 11 clinical relevant antibiotics and Omp35, Omp36 porins from Enterobacter aerogenes, that Omp35 has generally higher antibiotic-porin interaction than Omp36. Interestingly, E. aerogenes preferentially expresses Omp36 in human hosts. These in vitro results also correlate well with in vivo data collected from bacterial viability assays.On the other side, we studied the effect of magnesium on the antibiotic-porin interactions in order to approach physiological conditions. Since fluoroquinolones antibiotics have relatively heavy dipole, they can be used as probes to detect the electrostatic properties at the constriction zone of the porin channels. We found that the addition of magnesium strengthened the interaction between norfloxacin antibiotics and Omp36. The results indicate that the electrostatic properties of the constriction zone are altered and show a more efficient route to transport the norfloxacin.In conclusion, the porin properties can be better understood when considering the host environment. Improved understanding of antibiotic-porin interaction kinetics and the correlated antibiotic translocation based cell killing would facilitate the understanding on porin specificity thus the design of new antibiotics that can more efficiently reach high intracellular concentration.

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.

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 automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.