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.
Neurogen Corporation, Branford, CT 06405, USA. T.Knight@Cerebrus.ltd.uk
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Knight
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Hartnett
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Marks
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Brown
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Gallager
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Tallman
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Ramabhadran
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D011487:Protein Conformation; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D018411:Spodoptera
D011963:Receptors, GABA-A; D011994:Recombinant Proteins