Stable expression of human GABAC receptor ρ1 subunit in a human embryonic kidney cell line

Garry R Cutting, J. Mickle, C. J. Blaschak, A. S. Hackam

Research output: Contribution to journalArticle

Abstract

Purpose. Human GABA receptor subunits ρ1 and ρ2 form homooligomeric GABA-gated chloride channels with properties similar to the GABA type C receptors identified in retina. Characterization of ρ1 and ρ2 has relied primarily on recordings of injected oocytes. As the intracellular trafficking and folding of mammalian proteins, and consequently their functional properties, can differ between this expression system and mammalian cells, we created a human cell line stably expressing human ρ1. Methods. ρ1 cDNA tagged with the synthetic epitope FLAG(DYKDDDDK) (ρ1-FLAG, which has channel properties indistinguishable from native ρ1), was ligated into the mammalian expression vector pBK-RSV (Stratagene). 5 × 105 HEK 293 cells (ATCC#1573) were transfected with a DNA-lipofectin (BRL) mixture and selected on G418 (400 μg/ml) 72 hours after transfection. Twenty-four G418-resistant colonies were picked and expanded for analysis. Genomic DNA and total RNA extraction was by Trizol reagent (BRL), and hybridization to ρ1-specific sequences was by standard procedures. Whole-cell patch-clamp was performed on the stable and control cell lines at 37°C using a voltage-clamp protocol with a holding potential of -60mV. Results. Southern blotting with ρ1 probes confirmed genomic integration of full-length ρ1-FLAG in 6 out of 6 lines analyzed. Northern blotting, using β-actin as an internal standard, indicated that ρ1-FLAG expression varied between each of the 6 lines, whereas non-transfected cells had no ρ1 hybridizing signal. The highest expressing line was chosen for patch-clamp analysis. Current increased 15% (approximately 100 pA) in this stable cell line upon addition of 5 μM GABA (n=3). These currents were fully inhibited by the channel blocker picrotoxin (500 μM), but not by bicuculline (500 μM). Conclusion. We have demonstrated by DNA, RNA and functional analyses the establishment of a HEK 293 line stably expressing ρ1-FLAG. Preliminary pharmacological characterization of these cells indicates the presence of novel bicuclline-insensitive receptors that are inhibited by picrotoxin. This cell line will serve as a model system to assess the association between ρ1 receptors and GABAC receptors identified in retina, and will also be useful for biogenesis studies of the ρ1 receptor.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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Kidney
Cell Line
gamma-Aminobutyric Acid
Retina
DNA
RNA
Picrotoxin
Chloride Channels
GABA Receptors
Bicuculline
HEK293 Cells
Protein Folding
Southern Blotting
Northern Blotting
Oocytes
Transfection
Actins
Epitopes
Complementary DNA
Pharmacology

ASJC Scopus subject areas

  • Ophthalmology

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Stable expression of human GABAC receptor ρ1 subunit in a human embryonic kidney cell line. / Cutting, Garry R; Mickle, J.; Blaschak, C. J.; Hackam, A. S.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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title = "Stable expression of human GABAC receptor ρ1 subunit in a human embryonic kidney cell line",
abstract = "Purpose. Human GABA receptor subunits ρ1 and ρ2 form homooligomeric GABA-gated chloride channels with properties similar to the GABA type C receptors identified in retina. Characterization of ρ1 and ρ2 has relied primarily on recordings of injected oocytes. As the intracellular trafficking and folding of mammalian proteins, and consequently their functional properties, can differ between this expression system and mammalian cells, we created a human cell line stably expressing human ρ1. Methods. ρ1 cDNA tagged with the synthetic epitope FLAG(DYKDDDDK) (ρ1-FLAG, which has channel properties indistinguishable from native ρ1), was ligated into the mammalian expression vector pBK-RSV (Stratagene). 5 × 105 HEK 293 cells (ATCC#1573) were transfected with a DNA-lipofectin (BRL) mixture and selected on G418 (400 μg/ml) 72 hours after transfection. Twenty-four G418-resistant colonies were picked and expanded for analysis. Genomic DNA and total RNA extraction was by Trizol reagent (BRL), and hybridization to ρ1-specific sequences was by standard procedures. Whole-cell patch-clamp was performed on the stable and control cell lines at 37°C using a voltage-clamp protocol with a holding potential of -60mV. Results. Southern blotting with ρ1 probes confirmed genomic integration of full-length ρ1-FLAG in 6 out of 6 lines analyzed. Northern blotting, using β-actin as an internal standard, indicated that ρ1-FLAG expression varied between each of the 6 lines, whereas non-transfected cells had no ρ1 hybridizing signal. The highest expressing line was chosen for patch-clamp analysis. Current increased 15{\%} (approximately 100 pA) in this stable cell line upon addition of 5 μM GABA (n=3). These currents were fully inhibited by the channel blocker picrotoxin (500 μM), but not by bicuculline (500 μM). Conclusion. We have demonstrated by DNA, RNA and functional analyses the establishment of a HEK 293 line stably expressing ρ1-FLAG. Preliminary pharmacological characterization of these cells indicates the presence of novel bicuclline-insensitive receptors that are inhibited by picrotoxin. This cell line will serve as a model system to assess the association between ρ1 receptors and GABAC receptors identified in retina, and will also be useful for biogenesis studies of the ρ1 receptor.",
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T1 - Stable expression of human GABAC receptor ρ1 subunit in a human embryonic kidney cell line

AU - Cutting, Garry R

AU - Mickle, J.

AU - Blaschak, C. J.

AU - Hackam, A. S.

PY - 1996/2/15

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N2 - Purpose. Human GABA receptor subunits ρ1 and ρ2 form homooligomeric GABA-gated chloride channels with properties similar to the GABA type C receptors identified in retina. Characterization of ρ1 and ρ2 has relied primarily on recordings of injected oocytes. As the intracellular trafficking and folding of mammalian proteins, and consequently their functional properties, can differ between this expression system and mammalian cells, we created a human cell line stably expressing human ρ1. Methods. ρ1 cDNA tagged with the synthetic epitope FLAG(DYKDDDDK) (ρ1-FLAG, which has channel properties indistinguishable from native ρ1), was ligated into the mammalian expression vector pBK-RSV (Stratagene). 5 × 105 HEK 293 cells (ATCC#1573) were transfected with a DNA-lipofectin (BRL) mixture and selected on G418 (400 μg/ml) 72 hours after transfection. Twenty-four G418-resistant colonies were picked and expanded for analysis. Genomic DNA and total RNA extraction was by Trizol reagent (BRL), and hybridization to ρ1-specific sequences was by standard procedures. Whole-cell patch-clamp was performed on the stable and control cell lines at 37°C using a voltage-clamp protocol with a holding potential of -60mV. Results. Southern blotting with ρ1 probes confirmed genomic integration of full-length ρ1-FLAG in 6 out of 6 lines analyzed. Northern blotting, using β-actin as an internal standard, indicated that ρ1-FLAG expression varied between each of the 6 lines, whereas non-transfected cells had no ρ1 hybridizing signal. The highest expressing line was chosen for patch-clamp analysis. Current increased 15% (approximately 100 pA) in this stable cell line upon addition of 5 μM GABA (n=3). These currents were fully inhibited by the channel blocker picrotoxin (500 μM), but not by bicuculline (500 μM). Conclusion. We have demonstrated by DNA, RNA and functional analyses the establishment of a HEK 293 line stably expressing ρ1-FLAG. Preliminary pharmacological characterization of these cells indicates the presence of novel bicuclline-insensitive receptors that are inhibited by picrotoxin. This cell line will serve as a model system to assess the association between ρ1 receptors and GABAC receptors identified in retina, and will also be useful for biogenesis studies of the ρ1 receptor.

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