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Establishment of Cell-Free Electrophysiology for Ion Transporters: Application for Pharmacological Profiling

Nicolas Flores-Herr

Thomas Licher

Lead Identification Technologies, New Assay Technologies, Sanofi-Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany.

Henning Vollert

Lead Identification Technologies, New Assay Technologies, Sanofi-Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany; Industriepark Hoechst Building H812 D-65926 Frankfurt am Main, Germany henning.vollert{at}sanofi-aventis.com

Ion transporters are emerging targets of increasing importance to the pharmaceutical industry because of their relevance to a wide range of numerous indications of cardiovascular, metabolic, and inflammatory diseases. However, traditional iontransporter assay technologies using radioactive or fluorescent ligands and substrates or manual patch clamping suffer from several problems: limited sensitivity and robustness, significant numbers of false positives and false negatives, and cost. The authors describe a novel method for the measurement of ion transporters using cell-free electrophysiology based on the SURFE ² R (surface electrogenic event reader) technology platform. The main advantages of the method described here are high sensitivity and simple handling. Material for assays is mainly a simplemembrane preparation, which can be stored over weeks and months. Thus, the application of the method does not depend on a permanently running cell-culture lab. The application of the technology itself uses a bench-top system and chips loaded with membrane fragments. The SURFE ² R technology was used to establish an Na+/Ca2+-exchanger assay. The assay performance, as judged by the Z' value of 0.73 and the signal-to-background ratio of 7.6, suggests that this is a reliable and robust assay. The authors compared the technology with patch-clamp experiments: Themeasurement of activity of 17 different inhibitors and the determination of an IC ₅₀value indicated a good correlation between SURFE ² R technology and patch clamp results. Using the SURFE ² R technology, results were obtainedwith 20 times higher throughput and required less-qualified personnel comparedwith manual patch clamping.

Key Words: ion transporters • cell-free electrophysiology • sodium-calcium exchanger • patch clamp • solid-supported membrane

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