Molecular size of recombinant alpha1beta1 and alpha1beta1gamma2 GABAA receptors expressed in Sf9 cells

The present study examines the physical properties of recombinant human GABAA receptors. The baculovirus/Sf9 cell system was used to express combinations of human GABAA receptor subunits: alpha 1 alone, alpha 1 with beta 1, and alpha 1 with beta 1 and gamma 2. Receptors were solubilized using 1% Triton X-100. In sucrose density gradients containing 150mM NaCl, alpha 1 beta 1 receptor-detergent complexes sedimented more slowly than alpha 1 beta 1 gamma 2 constructs (sedimentation coefficient = 7.00 +/- 0.32 and 8.63 +/- 0.48 S, respectively). Stokes' radii for the two receptor-detergent complexes were determined by gel filtration in Sephacryl S-300. These experiments were performed in the presence of 1 M sodium chloride to prevent aggregation. The Stokes' radii for alpha 1 beta 1 and alpha 1 beta 1 gamma 2 receptor-detergent complexes were 9.06 +/- 0.23 and 7.91 +/- 0.19 nm, respectively. Sedimentation experiments in 1 M NaCl revealed similar sedimentation coefficients for alpha 1 beta 1 and alpha 1 beta 1 gamma 2 receptor-detergent complexes (8.79 +/- 0.59 and 8.46 +/- 0.72 S, respectively). The molecular weight of the alpha 1 beta 1 receptor excluding detergent was estimated to be 281 +/- 19 kDa, that of the alpha 1 beta 1 gamma 2 receptor, 247 +/- 21 kDa. This difference is not statistically significant. Given subunit molecular weights which are close to 50 kDa, this suggested a pentameric structure for the majority of alpha 1 beta 1 gamma 2 receptors, and that alpha 1 beta 1 receptors are not"assembly intermediates" with fewer subunits.