Development of a Novel Automated Ion Channel Recording Method Using "Inside-Out" Whole-Cell MembranesDiscovery Neuroscience, Wyeth Research
Wyeth Bio Medical Engineering, Princeton, NJ
Discovery Neuroscience, Wyeth Research Efforts to develop novelmethods for recording from ion channels have been receiving increased attention in recent years. In this study, the authors report a unique "inside-out" whole-cell configuration of patch-clamp recording that has been developed. This method entails adding cells into a standard patch pipette and, with positive pressure, obtaining a gigaseal recording from a cell at the inside tip of the electrode. In this configuration, the cellmay be moved through the air, first rupturing part of the cellularmembrane and enabling bath access to the intracellular side of the membrane, and then into a series of wells containing differing solutions, enabling robotic control of all the steps in an experiment. The robotic system developed here fully automates the electrophysiological experiments, including gigaseal formation, obtaining whole-cell configuration, data acquisition, and drug application. Proof-of-principle experiments consisting of application of intracellularly acting potassium channel blockers to K+ channel cell lines resulted in a very rapid block, aswell as block reversal, of the current. This technique allows compound application directly to the intracellular side of ion channels and enables the dissociation of compound inactivities due to cellular barrier limitations. This technique should allow for parallel implementation of recording pipettes and the future development of larger array-based screening methods.
Key Words: potassium channels • screening • patch clamp • intracellular
This version was published on December
1, 2005 Journal of Biomolecular Screening, Vol. 10, No. 8,
806-813 (2005) |
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