title: Expression of functional GABAA receptors in transfected L929 cells isolated by immunomagnetic bead separation.
abstract: Transient cotransfection of fibroblasts, with plasmids encoding individual GABAA receptor (GABAAR) subunits, has provided a model to characterize the pharmacological and kinetic properties of receptor subtype combinations. However, identifying transfected cells for electrophysiological recording is often difficult due to low transfection efficiencies. Selection of transfected cells has required cotransfection with a marker gene and fluorescence microscopic localization prior to recording. To circumvent these problems, two GABAAR subtypes combinations in transfected L929 cells were isolated with a novel biomagnetic separation system. Cell selection was accomplished by cotransfection with a plasmid (pHook-1) encoding a single-stranded cell surface antibody (sFv), which bound to ferromagnetic beads, coated with an antigen (phOx). Bead-covered cells were then magnetically separated from non-transfected cells. Bead-selected cells cotransfected with alpha 6, beta 3 and gamma 2L subtypes, expressed GABAAR currents in 95% (41/43) of cells recorded. Cells cotransfected with alpha 5, beta 3 and gamma 2L subtypes had an EC50 for GABA of 5.4 microM and a Hill slope of 1.4. Membrane patches from cells expressing the alpha 5 beta 3 gamma 2L isoform demonstrated single channel currents with a main conductance state of 23 pS. Magnetic bead immunoselection provides a purified population of transfected cells well suited for whole cell and single channel recording.
authors.affiliation: Department of Neurology, University of Michigan Medical Center, Ann Arbor, USA.
authors.forename: L J
authors.initials: LJ
authors.lastname: Greenfield
authors.forename: F
authors.initials: F
authors.lastname: Sun
authors.forename: T R
authors.initials: TR
authors.lastname: Neelands
authors.forename: E C
authors.initials: EC
authors.lastname: Burgard
authors.forename: J L
authors.initials: JL
authors.lastname: Donnelly
authors.forename: R L
authors.initials: RL
authors.lastname: MacDonald
mesh_terms: D000818:Animals; D002460:Cell Line; D018189:Immunomagnetic Separation / Q000379:methods*; D051379:Mice; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000187:drug effects / Q000276:immunology*; D014162:Transfection / Q000379:methods; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid