Figure 3. Functional analysis of GABA_B(1b) glycosylation mutants.

(A) Western blot. WT, wild-type GABA_B(1b); lanes 2–6, single-point mutants; Δglyc, GABA_B(1b)Δglyc, with all glycosylation sites mutated. No molecular mass shifts are observed between single-point glycosylation mutants; molecular mass was estimated to be approx. 130–135 kDa. The calculated molecular mass of GABA_B(1b)Δglyc plus EGFP is 122.5 kDa. (B) Surface expression of glycosylation-deficient GABA_B(1b) receptor. Staining for the haemagglutinin tag (first row, HA-staining), EGFP emission (second row, EGFP-Em) and overlap of the two (third row, overlap). (C) Fluorimetric analysis. HEK-293 cells, electrophorated with the cDNAs of GABA_B(2), chimaeric G_αqzIC, and either GABA_B(1b) wild-type (a) or mutants (b–h) were perfused with 0.1 mM GABA for 60 s. The generated response could be completely antagonized using 10 μM CGP54626A (grey segment on the timescale), since no response was measured by subsequent application of GABA (black segment on the right side of timescale, 60 s). Lanes (a–g) display changes in fluorescence in response to GABA application 24 h post-transfection. Lanes (g) and (h) correspond to the GABA_B(1b)Δglyc form, lane (g) 24 h after transfection, lane (h) 48 h after transfection. (D) Agonist concentration–response curves of wild-type and glycosylation-deficient GABA_B(1b) receptors. HEK-293 cells were measured in FLIPR [12], 48 h after transfection of each variant together with GABA_B(2) and chimaeric G_αqzIC. Data points show average fluorescence values of 4 (WT) or 8 (Δglyc) wells ±S.E.M., normalized to the average response to 0.1 mM GABA. Hill equation fits gave EC₅₀ values of 2.4 (1.7, 3.4) for WT and 6.4 μM (5.3, 7.8) for the Δglyc mutant (mean, 95% confidence interval).