Development of a novel automated ion channel recording method using "inside-out" whole-cell membranes.
Efforts to develop novel methods 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 cell may be moved through the air, first rupturing part of the cellular membrane 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, as well 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 in activities 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.
Vasilyev
Dmitry V
DV
Discovery Neuroscience, Wyeth Research, Princeton, NJ 08543-8000, USA.
Merrill
Thomas L
TL
Bowlby
Mark R
MR
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004594:Electrophysiology / Q000295:instrumentation*; D007473:Ion Channels / Q000502:physiology; D008566:Membranes / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000295:instrumentation* / Q000379:methods; D012371:Robotics
D007473:Ion Channels