Whole-cell and single-channel alpha1 beta1 gamma2S GABAA receptor currents elicited by a "multipuffer" drug application device.
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.
Greenfield
L J
LJ
Departments of Neurology, Neuroscience Laboratory Building, 1103 E. Huron, Ann Arbor MI 48904-1687, USA.
Macdonald
R L
RL
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
D018118:Chloride Channels; D007473:Ion Channels; D004364:Pharmaceutical Preparations; D018079:Receptors, GABA; D011994:Recombinant Proteins; D012996:Solutions; D005680:gamma-Aminobutyric Acid; D011188:Potassium