ASSEMBLY OF GABA(A) RECEPTORS COMPOSED OF ALPHA-1 AND BETA-2 SUBUNITSIN BOTH CULTURED NEURONS AND FIBROBLASTS

GABA(A) receptors are believed to be pentameric heterooligomers, which can be constructed from six subunits (alpha, beta, gamma, delta, epsi lon, and rho) with multiple members, generating a large potential for receptor heterogeneity. The mechanisms used by neurons to control the assembly of these receptors, however, remain unresolved. Using Semliki Forest virus expression we have analyzed the assembly of 9E10 epitope -tagged receptors comprising alpha 1 and beta 2 subunits in baby hamst er kidney cells and cultured superior cervical ganglia neurons. Homome ric subunits were retained within the endoplasmic reticulum, whereas h eteromeric receptors were able to access the cell surface in both cell types. Sucrose density gradient fractionation demonstrated that the h omomeric subunits were incapable of oligomerization, exhibiting 5 S se dimentation coefficients. Pulse-chase analysis revealed that homomers were degraded. With half-lives of similar to 2 hr for both the alpha 1 ((9E10)) and beta 2((9E10)) subunits. Oligomerization of the alpha 1(( 9E10)) and beta 2((9E10)) subunits was evident, as demonstrated by the formation of a stable 9 S complex, but this process seemed inefficien t. Interestingly the appearance of cell surface receptors was slow, la gging up to 6 hr after the formation of the 9 S receptor complex. Usin g metabolic labeling a ratio of alpha 1((9E10)):beta 2((9E10)) of 1:1 was found in this 9 S fraction. Together the results suggest that GABA (A) receptor assembly occurs by similar mechanisms in both cell types, with retention in the endoplasmic reticulum featuring as a major cont rol mechanism to prevent unassembled receptor subunits accessing the c ell surface.