State-of-the-Art Automated Patch Clamp Devices: Heat Activation, Action Potentials, and High Throughput in Ion Channel Screening

Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. The availability of standardized cell lines expressing ion channels of interest lead to the development of diverse automated patch clamp (APC) systems with high-throughput capabilities. These systems are now available for drug screening, but there are limitations in the application range. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. The addition of well controlled and fast solution exchange, temperature control and the availability of the current clamp mode are required to analyze standard cell lines and excitable cells such as stem cell-derived cardiomyocytes in a more physiologically relevant environment. Here we describe two systems with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike. The here utilized medium throughput APC device is a planar patch clamp system capable of recording up to eight cells simultaneously. Features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The high-throughput system used here is a planar patch clamp screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The data provided reveals the suitability and relevance of both APC platforms for drug discovery, ion channel research, and safety testing

Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport. Electrogenic co-transport of H+/glycylglycine (GlyGly) was observed across a pH range of 5.0 to 9.0. The pH dependence is described by a bell-shaped activity curve and two pK values. KM and relative Vmax values of various canonical and non-canonical peptide substrates were contextualized with current mechanistic understandings of hPepT1. Finally, specific inhibition was observed for various inhibitors in a high throughput format, and IC50 values are reported. Taken together, these findings contribute to promoting the design and analysis of pharmacologically relevant substances

Coupled impedance & field potential data analysis of in vitro cardiomyocyte assays

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A novel gain-of-function mutation of Piezo1 is functionally affirmed in red blood cells by high-throughput patch clamp

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Bilayer-spanning DNA nanopores with voltage-switching between open and closed state.

Membrane-spanning nanopores from folded DNA are a recent example of biomimetic man-made nanostructures that can open up applications in biosensing, drug delivery, and nanofluidics. In this report, we generate a DNA nanopore based on the archetypal six-helix-bundle architecture and systematically characterize it via single-channel current recordings to address several fundamental scientific questions in this emerging field. We establish that the DNA pores exhibit two voltage-dependent conductance states. Low transmembrane voltages favor a stable high-conductance level, which corresponds to an unobstructed DNA pore. The expected inner width of the open channel is confirmed by measuring the conductance change as a function of poly(ethylene glycol) (PEG) size, whereby smaller PEGs are assumed to enter the pore. PEG sizing also clarifies that the main ion-conducting path runs through the membrane-spanning channel lumen as opposed to any proposed gap between the outer pore wall and the lipid bilayer. At higher voltages, the channel shows a main low-conductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation. This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes. These findings settle a discrepancy between two previously published conductances. By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.The SH lab is supported by the Leverhulme Trust (RPG-170), UCL Chemistry, EPSRC (Institutional Sponsorship Award), the National Physical Laboratory, and Oxford Nanopore Technologies. KG acknowledges funding from the Winton Program of Physics for Sustainability, Gates Cambridge and the Oppenheimer Trust. UFK was supported by an ERC starting grant #261101.This is the final version of the article. It was first published by ACS under the ACS AuthorChoice license at http://dx.doi.org/10.1021/nn5039433 This permits copying and redistribution of the article or any adaptations for non-commercial purposes

Constructing semantic representations using the MDL principle

Words receive a significant part of their meaning from use in communicative settings. The formal mechanisms of lexical acquisition, as they apply to rich situational settings,may also be studied in the limited case of corpora of written texts. This work constitutes an approach to deriving semantic representations for lexemes using techniques from statistical induction. In particular, a number of variations on the MDL principle were applied to selected sample sets and their influence on emerging theories of word meaning explored. We found that by changing the definition of description length for data and theory - which is equivalent to different encodings of data and theory - we may customize the emerging theory, augmenting and altering frequency effects. Also the influence of the stochastic properties of the size of the theory has been demonstrated. The results consist in a set of distributional properties of lexemes, which reflect cognitive distinctions in the meaning of words