SITE-DIRECTED MUTAGENESIS OF N-LINKED GLYCOSYLATION SITES ON THE GAMMA-AMINOBUTYRIC-ACID TYPE-A RECEPTOR ALPHA-1 SUBUNIT

Oligonucleotide-directed mutagenesis was used to mutate the two potent ial sites for N-linked glycosylation on the rat gamma-aminobutyric aci d (GABA)(A) receptor alpha 1 subunit. Wild-type (WT) or mutant alpha 1 subunits [asparagine to glutamine substitutions at position 10 (alpha 1Q(10)), 110 (alpha 1Q(110)), or both 10 and 110 (alpha 1Q(10/110))] were coexpressed with beta 1 and gamma 2 subunits in Xenopus oocytes. Removal of either one or both potential sites for N-linked glycosylati on resulted in expression, in Xenopus oocytes, of functional GABA(A) r eceptors with pharmacological properties similar to those observed for the WT receptor. WT and mutant alpha 1 subunits were co-transfected w ith beta 1 and gamma 2 subunits in human embryonic kidney 293 cells. W T and mutant alpha 1 subunits expressed in 293 cells were photoaffinit y labeled with [H-3]flunitrazepam. Co-transfection of alpha 1WT, alpha 1Q(10), or alpha 1Q(110) subunits in combination with beta 1 and gamm a 2 GABA(A) receptor subunits resulted in the labeling of single bands , with approximate molecular masses of 54, 49, and 50 kDa, respectivel y. The decrease in molecular mass for both the alpha 1Q(10) and alpha 1Q(110) mutants suggests that both consensus sequences for N-linked gl ycosylation are used in 293 cells. Low levels of [H-3]flunitrazepam bi nding prevented visualization of the alpha 1Q(10/110) double mutant. T he 293 cells transfected with either the alpha 1Q(10) or alpha 1Q(110) mutant in combination with beta 1 and gamma 2 subunits expressed sign ificantly lower levels of [H-3]Ro15-1788 binding, relative to WT level s. In addition, [H-3]Ro15-1788 binding was undetectable in 293 cells e xpressing the alpha 1Q(10/110) double mutant. When transfected 293 cel ls were grown at 30 degrees, [H-3]Ro15-1788 binding to alpha 1Q(10) an d alpha 1Q(110) GABA(A) receptors was restored to levels comparable to that for WT receptors. [H-3]Ro15-1788 binding to alpha 1Q(10/110) was not reliably detected at 30 degrees. Similar results were observed us ing [H-3]muscimol. These data suggest that intracellular processing an d transport of the glycosylation-deficient GABA(A) receptor alpha 1 su bunit is temperature sensitive. Furthermore, the observed differences between the two expression systems may be accounted for by the typical ly lower temperature used for maintaining microinjected Xenopus oocyte s. Thus, although glycosylation is not an absolute requirement for GAB A(A) receptor expression, it has a profound effect on the processing o f at least the al receptor and its subsequent assembly into a mature r eceptor.