Figure 2. Expression, photolabelling with [¹²⁵I]CGP71872 and agonist activation of GABA_B(1b)ECD deletion mutants.

(A) Western blot. The immunoblot was performed with antibodies specific for GABA_B(1) subunits, to confirm the expression of the designed mutants. Protein concentrations were estimated (Biochronic assay; Pierce) and an equal amount of protein was loaded per lane. Lane 1, indicated with a minus, shows membranes of cells transfected without cDNA. Lane WT, wild-type GABA_B(1b). Since proteins (molecular mass 100 kDa) are expressed as EGFP fusion proteins, their molecular mass is 20 kDa higher. A lower band is also observed which results from degradation of the full-length protein. This degraded form is detected by the antibody, but is not able to bind the antagonist (see Figure 2B). (B) Photolabelling. Samples identical with those detected in Figure 2(A) were tested for their ability to bind the highly specific GABA_B(1) inhibitor [¹²⁵I]CGP71872. WT indicates wild-type GABA_B(1b), lanes GB1bΔ42, GB1bΔ49, GB1bΔ55 and GB1bΔ60 indicate the N-terminally truncated receptors; lanes GB1bΔ454, GB1bΔ443 and GB1bΔ433 indicate GABA_B(1b) receptors with C-terminal deletions of the ECD. Lane GB1bΔ55/Δ443 shows GABA_B(1b) receptors containing deletions both at the N- and C-terminus of the ECD. No cross-reaction between the iodinated antagonist and HEK-293 untransfected cells was observed (first lane). (C) HEK-293 cells, electrophorated with cDNAs of GABA_B(2), chimaeric G_αqzIC, GABA_B(1b) wild-type (a) and mutants (b–h) were perfused with 0.1 mM GABA for 60 s (short horizontal line on the left side of the 13 min timescale at the bottom of the picture). Changes in intracellular calcium concentration [Ca²⁺]_i were monitored by measuring the ratio of fura 2/AM fluorescence (510 nm) excited at 340 and 380 nm. The generated response could be completely antagonized by preapplication of 10 μM CGP 54626A (short black and grey horizontal lines at the right bottom of the picture). Lanes a–h display changes in fluorescence in response to GABA and CGP54626A application.