Fig 2: GABABR structure. (A) Multiple sequence alignment showing the conserved Arginine residue at position 212 (highlighted in red) across various species. (B) GABABR (pdb:7 EB2) bound to G protein embedded in a phospholipid membrane, with the GB2 variants highlighted in red.

Fig 3: Functional characterization of GB1/2R212Q and GB1/2R212W receptors in heterologous cells. (A,B) Cell surface and total Myc-GB2 expression of GB1/2, GB1/2R212Q (A) and GB1/2R212W (B) receptors in transfected HEK293T cells. GB1 and Myc-GB2 subunits were co-expressed and the surface GB2 FIGURE 5 (Continued)immunofluorescence assessed in living cells using anti-Myc and AF488-conjugated secondary antibodies. Total GB2 immunofluorescence was determined with anti-GB2 and secondary AF647-conjugated antibodies after cell permeabilization. Scale bar: 10 μm. (C,D) Bar graphs showing the GB2, GB1/2R212Q (C) and GB1/2R212W (D) surface-to-total immunofluorescence ratios. GB1/2 (100.00 ± 2.42% n = 78), GB1/2R212Q (79.70 ± 3.25% n = 84), unpaired t-test with Welch’s correction; GB1/2 (100 ± 3.11% n = 128), GB1/2R212W (96.42 ± 3.03% n = 107), Mann–Whitney test. (E) Representative immunoblots of HEK293T cells expressing GB1/2, GB1/2R212Q, and GB1/2R212W. Myc-GB2 was detected with the anti-Myc antibody. (F) Total expression of GB1/2, GB1/2R212Q, and GB1/2R212W in HEK293T cells. GB1/2 (2.46 ± 0.14 n = 6), GB1/2R212Q (2.47 ± 0.17 n = 6); GB1/2 (2.51 ± 0.28 n = 5); GB1/2R212W (2.18 ± 0.17 n = 5), unpaired t-test. (G) Concentration-response curves depicting GABA-induced luciferase activity (sreLuc bioluminescence) of GB1/2 and GBR1/2R212Q in transfected HEK293T cells. GABA concentration-response curves were fitted using a three-parameter log (agonist) vs. response nonlinear regression curve. n = 15 experiments for each curve. (H) Same as in (G) for GBR1/2R212W. n = 8 experiments for each curve. (I) Bar graphs of constitutive activity, Emax and EC50 values for GABA concentration–response curves at GB1/2, GB1/2R212Q and GB1/2 + GB1/2R212Q receptors. ANOVA with Tukey’s post-hoc analysis (constitutive activity, EMax), and Kruskal-Wallis with Dunn’s post-hoc (EC50). (J) Same as in (I) for GBR1/2R212W receptors. ANOVA with Dunnett’s T3 post-hoc (constitutive activity), Kruskal-Wallis with Dunn’s post-hoc (Emax), and ANOVA with Tukey’s post-hoc (EC50). All data are mean ± SEM. p-values are given in the bar graphs.

Fig 4: VFTD dynamics of GABBR2 p.Arg212Gln and p.Arg212Trp in active and/or inactive states (pdb:7 EB2,7C7S). (A) Illustration of the inactive structure (pdb: 7C7S) of GABBR2 p.Arg212Gln. We monitored the distance between GB1-LBupper (the center of mass of Cα for residues 222–235 and 247–260; green) and GB1-LBlower (the center of mass of Cα for residues 347–358 and 368–382; light green). (B) Distance in angstrom between GB1-LBupper and GB1-LBlower in the active structure (pdb:7 EB2) throughout the simulation. p.Arg212Trp was not simulated in the active structure. (C) The distance between GB1-LBupper and GB1-LBlower in the inactive structure (pdb: 7C7S). GABBR2 p.Arg212Gln and p.Arg212Trp exhibit lower values than those of the WT receptor, closer to those observed in the active structure of the WT receptor.

Fig 5: GABBR2 p.Arg212Gln and p.Arg212Trp TM segment distances. (A) The distance between centers of mass of TM6 helices (805–825 in GB1, 692–712 in GB2) segments over time. (B,C) Illustration demonstrating that TM6 helices are closer together in the inactive structure of p.Arg212Gln than in WT receptor, while TM6 helices are further apart in p.Arg212Trp than in WT receptor. (D) The distance between centers of mass of TM5 helices (769–789 in GB1, 655–675 in GB2) segments over time. (E,F) In the inactive structures of p.Arg212Gln receptors, TM5 helices exhibit a greater separation compared to their positioning in WT receptors. Conversely, in p.Arg212Trp receptors, TM5 helices are observed to be closer together than in the WT receptor structures. (G) The distance between centers of mass of TM4 helices (711–731 in GB1, 598–618 in GB2) segments over time. (H,I) In the inactive structure of the WT receptor, TM4 helices are closer together than in the inactive structure of the p.Arg212Gln variant. The values for p.Arg212Trp closely resemble those observed for the WT receptor. Additional distance comparisons for TM1-3 and TM7 are shown in Supplementary Figure S2. GB1 is in red, GB2 is in blue. Darker shades represent GB2 variants. Lighter shades represent the GB2 protein. The GB2 protein segment is shown in cyan, and the GABBR2 p.Arg212Gln\Trp segment in pink.