A novel method for patch-clamp automation

An increasing demand of the pharmaceutical industry for automated electrophysiological stations for ion channel drug discovery has recently resulted in the development of several commercial platforms for secondary and safety screening of ion channel modulators. These commercial systems have demonstrated an enhanced throughput, however, often at the expense of some quality-sensitive aspects of traditional patch-clamp recordings. To improve data quality and content, we have developed a patch-clamp robot that fully automates manual patch-clamp recordings, including patch pipette handling, gigaseal formation, obtaining whole-cell or perforated-cell configuration, drug application, and data acquisition. Utilization of glass micropipettes results in high-quality electrophysiological recordings with an overall success rate of about 30% in perforated-cell mode. A fast drug application system with low volume requirements (1–1.5 ml) allows the study of ligand-gated ion channels on a millisecond scale. As proof-of-concept, we present two assays developed for voltage-gated human ether-a-go-go-related and ligand-gated α7 nicotinic receptor ion channels. The system throughput was a single concentration–response curve every 30–40 min or 12–17 6-point concentration–response curves daily, representing a significant improvement of typical manual patch-clamp throughput. This system represents an efficient method for patch-clamp automation without the need for a complex and expensive electrophysiological set-up for cell visualization.

[1]  Peter Fromherz,et al.  Recombinant maxi-K channels on transistor, a prototype of iono-electronic interfacing , 2001, Nature Biotechnology.

[2]  G. Terstappen,et al.  Differential inhibition of rat alpha3* and alpha7 nicotinic acetylcholine receptors by tetrandrine and closely related bis-benzylisoquinoline derivatives. , 2005, Neuroscience letters.

[3]  Huimin Tao,et al.  Automated tight seal electrophysiology for assessing the potential hERG liability of pharmaceutical compounds. , 2004, Assay and drug development technologies.

[4]  Paul B Bennett,et al.  High throughput ion-channel pharmacology: planar-array-based voltage clamp. , 2003, Assay and drug development technologies.

[5]  Y. Kuo,et al.  Functional Properties of Homomeric, Human α7-Nicotinic Acetylcholine Receptors Heterologously Expressed in the SH-EP1 Human Epithelial Cell Line , 2003, Journal of Pharmacology and Experimental Therapeutics.

[6]  U. Warrior,et al.  Functional analysis of large conductance Ca2(+)-activated K(+) channels: ion flux studies by atomic absorption spectrometry. , 2003, Assay and drug development technologies.

[7]  M. Reed,et al.  Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells. , 2002, Biosensors & bioelectronics.

[8]  T J Campbell,et al.  Inhibition of HERG channels stably expressed in a mammalian cell line by the antianginal agent perhexiline maleate , 1999, British journal of pharmacology.

[9]  Christine Williams,et al.  Patch clamping by numbers. , 2004, Drug discovery today.

[10]  Xiaobo Wang,et al.  Automated electrophysiology: high throughput of art. , 2003, Assay and drug development technologies.

[11]  George M Whitesides,et al.  Microfabricated teflon membranes for low-noise recordings of ion channels in planar lipid bilayers. , 2003, Biophysical journal.

[12]  E. Sher.  Ion Channels in Drug Discovery and Development , 2003, Expert opinion on investigational drugs.

[13]  Robert H Blick,et al.  Whole cell patch clamp recording performed on a planar glass chip. , 2002, Biophysical journal.

[14]  Wei Zheng,et al.  High throughput assay technologies for ion channel drug discovery. , 2004, Assay and drug development technologies.

[15]  L. Mattheakis,et al.  Assay technologies for screening ion channel targets. , 2001, Current opinion in drug discovery & development.

[16]  Mark R Bowlby,et al.  Development of a Novel Automated Ion Channel Recording Method Using “Inside-Out” Whole-Cell Membranes , 2005, Journal of biomolecular screening.

[17]  Andreas Sewing,et al.  High-Throughput Screening for Ion Channel Modulators , 2002, Journal of biomolecular screening.

[18]  Sujatha M. Gopalakrishnan,et al.  Validation of FLIPR Membrane Potential Dye for High Throughput Screening of Potassium Channel Modulators , 2001, Journal of biomolecular screening.

[19]  O. Krishtal,et al.  Effect of internal fluoride and phosphate on membrane currents during intracellular dialysis of nerve cells , 1975, Nature.

[20]  G. Terstappen,et al.  Differential inhibition of rat α3* and α7 nicotinic acetylcholine receptors by tetrandrine and closely related bis-benzylisoquinoline derivatives , 2005, Neuroscience Letters.

[21]  F Bezanilla,et al.  Bilayer reconstitution of voltage-dependent ion channels using a microfabricated silicon chip. , 2001, Biophysical journal.

[23]  Rafael J. Taboryski,et al.  Upscaling and Automation of Electrophysiology: Toward High Throughput Screening in Ion Channel Drug Discovery , 2003 .

[24]  G. Terstappen,et al.  Functional analysis of native and recombinant ion channels using a high-capacity nonradioactive rubidium efflux assay. , 1999, Analytical biochemistry.

[25]  A Brüggemann,et al.  High quality ion channel analysis on a chip with the NPC technology. , 2003, Assay and drug development technologies.

[26]  A. Lepple-Wienhues,et al.  Flip the tip: an automated, high quality, cost-effective patch clamp screen. , 2003, Receptors & channels.

[27]  Alfred Stett,et al.  CYTOCENTERING: a novel technique enabling automated cell-by-cell patch clamping with the CYTOPATCH chip. , 2003, Receptors & channels.

[28]  D. Rothwarf,et al.  A benchmark study with sealchip planar patch-clamp technology. , 2003, Assay and drug development technologies.

[29]  B. Sakmann,et al.  Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches , 1981, Pflügers Archiv.

[30]  G. Terstappen.  Nonradioactive rubidium ion efflux assay and its applications in drug discovery and development. , 2004, Assay and drug development technologies.

[31]  A. Camm,et al.  Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. , 2003, Cardiovascular research.

[32]  Lei Wu,et al.  Ion-channel assay technologies: quo vadis? , 2001, Drug discovery today.

[33]  Soo Sen Lee,et al.  Flux assays in high throughput screening of ion channels in drug discovery. , 2003, Assay and drug development technologies.

[34]  Derek J Trezise,et al.  IonWorks™ HT: A New High-Throughput Electrophysiology Measurement Platform , 2003, Journal of biomolecular screening.