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 language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|Publication status||Published - Feb 15 1996|
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