Stable GABA(A) receptor intermediates in SF-9 cells expressing alpha1, beta2 and gamma2 subunits

Spodoptera frugiperda insect cells (Sf-9 cells) were used to study GABA(A) receptor assembly. Time courses of the expression level of alpha1beta2 and alpha1beta2gamma2 receptor protein showed [(3)H]muscimol binding to appear 2 hr before [(3)H]flunitrazepam and [(35)S]TBPS binding. This indicates that muscimol may bind to pentamers with an immature conformation or to molecules smaller than the pentamer. Binding studies performed on fractions from sucrose gradients loaded with solubilized alpha1beta2 or alpha1beta2gamma2 containing membranes revealed no binding other than to the pentameric fractions. Western blotting on fractionated sucrose gradients, however, clearly revealed the existence of GABA(A) receptor intermediates. The alpha1 subunit was seen in fractions corresponding to molecules smaller than the pentamer only when co-expressed with gamma2, indicating that the gamma2 subunit is needed for the alpha1 to form relatively long lasting intermediates. Moreover, Western blots revealed multiple isoforms for each subunit. In general, it was primarily the lower molecular weight forms that were detected in the pentameric fractions. The exception being for the alpha1 and gamma2 forms in subunit combinations that did not contain both of these subunits (i.e., alpha1, gamma2, alpha1beta2, beta2gamma2), where higher molecular weight forms were strongly represented. These findings show that alpha1 and gamma2 prefer specific protein forms when expressed together.