Expression of functional GABA(A) receptors in transfected L929 cells isolated by immunomagnetic bead separation

  • Greenfield L
  • Fang S
  • Neelands T
 et al. 
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Transient cotransfection of fibroblasts, with plasmids encoding individual GABA(A) receptor (GABA(A)R) 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 GABA(A)R subtype combinations in transfected L929 cells were isolated with a novel biomagnetic separation system. Cell selection was accomplished by cotransfection with a plasmid (pHook(TM)-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 α6, β3 and γ2L subtypes, expressed GABA(A)R currents in 95% (41/43) of cells recorded. Cells cotransfected with α5, β3 and γ2L subtypes had an EC50for GABA of 5.4 μM and a Hill slope of 1.4. Membrane patches from cells expressing the α5 β3 γ2L 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.

Author-supplied keywords

  • GABA(A) receptor
  • cDNA
  • electrophysiology
  • pHook
  • patch clamp
  • phOx antigen
  • sFv antibody
  • single channel
  • transfection

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  • L. J. Greenfield

  • Sun Fang

  • T. R. Neelands

  • E. C. Burgard

  • J. L. Donnelly

  • R. L. MacDonald

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