title: Whole-cell and single-channel alpha1 beta1 gamma2S GABAA receptor currents elicited by a "multipuffer" drug application device.
abstract: Pharmacological characterization of ion channels and receptors in cultured neurons or transfected cell lines requires microapplication of multiple drug solutions during electrophysiological recording. An ideal device could apply a large number of solutions to a limited area with rapid arrival and removal of drug solutions. We describe a novel "multipuffer" rapid application device, based on a modified T-tube with a nozzle made from a glass micropipette tip. Drug solutions are drawn via suction from open reservoirs mounted above the recording chamber through the device into a waste trap. Closure of a solenoid valve between the device and the waste trap causes flow of drug solution though the T-tube nozzle. Any number of drug solutions can be applied with rapid onset (50-100 ms) after a brief fixed delay (100-200 ms). Recombinant alpha1beta1gamma2S GABAA receptors (GABARs) transfected into L929 fibroblasts were recorded using whole-cell and single-channel configurations. Application of GABA resulted in chloride currents with an EC50 of 12.2 microM and a Hill slope of 1.27, suggesting more than one binding site for GABA. GABAR currents were enhanced by diazepam and pentobarbital and inhibited by bicuculline and picrotoxin. Single-channel recordings revealed a main conductance state of 26-28 pS. This device is particularly suitable for rapid, spatially controlled drug applications onto neurons or other cells recorded in the whole-cell configuration, but is also appropriate for isolated single-channel or multichannel membrane patch recordings.
authors.affiliation: Departments of Neurology, Neuroscience Laboratory Building, 1103 E. Huron, Ann Arbor MI 48904-1687, USA.
authors.forename: L J
authors.initials: LJ
authors.lastname: Greenfield
authors.forename: R L
authors.initials: RL
authors.lastname: Macdonald
mesh_terms: D000818:Animals; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology; D004553:Electric Conductivity; D004864:Equipment and Supplies; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D004364:Pharmaceutical Preparations / Q000008:administration & dosage*; D011188:Potassium / Q000008:administration & dosage / Q000494:pharmacology; D018079:Receptors, GABA / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins; D012996:Solutions; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000008:administration & dosage / Q000494:pharmacology
chemical_list: D018118:Chloride Channels; D007473:Ion Channels; D004364:Pharmaceutical Preparations; D018079:Receptors, GABA; D011994:Recombinant Proteins; D012996:Solutions; D005680:gamma-Aminobutyric Acid; D011188:Potassium