MOLECULAR-SIZE OF RECOMBINANT ALPHA(1)BETA(1) AND ALPHA(1)BETA(1)GAMMA(2) GABA(A) RECEPTORS EXPRESSED IN SF9 CELLS

The present study examines the physical properties of recombinant huma n GABA(A) receptors. The baculovirus/Sf9 cell system was used to expre ss combinations of human GABA(A) receptor subunits: alpha(1) alone, al pha(1) with, beta(1), and alpha(1) with beta(1) and gamma(2). Receptor s were solubilized using 1% Triton X-100. In sucrose density gradients containing 150mM NaCl, alpha(1)beta(1) receptor-detergent complexes s edimented more slowly than alpha(1)beta(1)gamma(2) constructs (sedimen tation coefficient=7.00+/-0.32 and 8.63+/-0.48S, respectively). Stokes ' radii for the two receptor-detergent complexes were determined by ge l filtration in Sephacryl S-300. These experiments were performed in t he presence of 1M sodium chloride to prevent aggregation. The Stokes' radii for alpha(1)beta(1) and alpha(1)beta(1)gamma(2) receptor-deterge nt complexes were 9.06 +/- 0.23 and 7,91 i 0,19 nm, respectively. Sedi mentation experiments in 1M NaCl revealed similar sedimentation coeffi cients for alpha(1)beta(1) and alpha(1)beta(1)gamma(2), receptor-deter gent complexes (8.79+/-0.59 and 8.46+/-0.72S, respectively). The molec ular weight of the alpha(1)beta(1) receptor excluding detergent was es timated to be 281 +/- 19 kDa, that of the alpha(1)beta(1)gamma(2) rece ptor, 247 +/- 21 kDa. This difference is not statistically significant . Given subunit molecular weights which are close to 50 kDa, this sugg ested a pentameric structure for the majority of alpha(1)beta(1)gamma( 2) receptors, and that alpha(1)beta(1) receptors are not ''assembly in termediates'' with fewer subunits.