Expression and purification of a functional heteromeric GABAA receptor for structural studies
Fig 9
Optimized mammalian expression parameters apply to other receptor GABAA receptor subtypes.
(A) A boost in ρ1-EGFP expression levels is observed at 30°C with addition of 10 mM sodium butyrate. (B) Similar to oocytes, the β subunit is required to form a homogeneous receptor in mammalian cells. In addition, the α1-EGFP/β1-LT receptor demonstrates higher expression levels than the α1-EGFP/β2-LT receptor. Expression for each experiment was performed at 30°C. For comparison, the fluorescence scale in (B) is approximately 2-fold greater than in (A). (C) Time course of α1-EGFP/β1-LT expression at 30°C (relative MOI = 0.6, α1:β1) followed by EGFP-FSEC suggests that the receptor demonstrates peak expression levels 60–72 hours post infection. (D) The α1-LT/β1-LT receptor displays GABA-gated current in oocytes by TEVC measurement.