GB1_ASRR, the GB1 subunit with mutation in the endoplasmic reticulum retention/retrieval signal RSRR, is functional in oocytes only when expressed with wt GB2 (A and B). In contrast, the double mutant GB1_AA/ASRR, which harbors an additional mutation in the dileucine endocytosis motif, is functional when coexpressed with either wt GB2 (C) or with GB2-CC, the coiled–coil mutant of GB2 (D). Thus, the C-terminal coiled–coil interaction of GB1 and GB2 is important for masking of both trafficking signals in the GB1 C terminus but does not play a role in the GIRK channel coupling of GABA_B receptors. The I–V plots from representative cells are shown.

All three GB2 intracellular loops are essential for GIRK channel coupling: coexpression of GB1 with individual intracellular loop chimeras GB2(1i1), GB2(1i2), and GB2(1i3) resulted in receptors without any functional activity, despite wt-like surface expression levels (E). The I–V plots from representative cells (A–C) and the summary plot (D) are shown. The surface expression of plasma membrane-expressed receptor complexes was determined in mammalian COS7 cells by using chemiluminescence-based assay (for details, see Materials and Methods). For these experiments, the extracellular domain of GB1 subunit was tagged with an HA epitope.

GB1 and GB2 C-terminal truncation mutants were functionally assayed by coexpression with GIRK1/GIRK2 channels in Xenopus oocytes. In this assay, binding of GABA to functional GABA_B receptors results in activation of inwardly rectifying K⁺ current; see Materials and Methods for experimental details. The I–V plots from representative cells (A–C) and the summary of all of the cells tested (D) are shown. In this and subsequent figures, GB1 subunit is schematically represented in black and GB2 subunit in gray; the notch in GB1 subunit represents the GABA-binding site. In all figures, summarized data (which were not normally distributed) are represented as statistical box charts (the horizontal lines in the box denote 25th, 50th, and 75th percentile values, the error bars denote 5th and 95th percentile values, the asterisks denote 1st and 99th percentile values, and the square symbol in the box denotes the mean). For summary plot and statistical analysis, currents were measured at −120 mV; GABA responses are expressed as a percentage of basal GIRK currents recorded in 40K solution just before GABA application. (A) Coexpression of the GB1 C-terminal truncation mutant GB1Δ102 with GB2 resulted in receptors indistinguishable from wt GABA_B receptors in their ability to activate GIRK channels. (B) Deletion of the GB2 C terminus in the GB1Δ102/GB2Δ195 receptor complex significantly attenuated its ability to mediate GIRK current activation (Kruskal–Wallis one-way ANOVA on ranks, followed by the Dunn's posttest). (C) GABA_B receptor function was preserved when the truncation of the GB2 C terminus was less severe, leaving intact the proximal 36 residues.

(A) GB1 intracellular loops can be exchanged with intracellular loops from GB2 [construct GB1(2i1–3)] without any effect on the GABA_B receptor function. (B) Similarly, replacement of GB1 loops with loops from mGluR1 [construct GB1(mG1i1–3)] results in fully functional receptors. mGluR1-derived sequences are represented in black outline. (C) Replacing the middle 11 residues in the GB1 i2 loop with a random coil peptide [construct GB1(i2xg11)] does not affect the receptor function. The sequence of i2 loop in GB1(i2xg11) is indicated, with replacement residues boxed in gray. The I–V plots from representative cells (A–C) and the summary plot (D) are shown.

GABA_B receptors are constitutive heterodimers. Subunit interactions include the noncovalent contacts between the N-terminal domains and the C-terminal coiled–coil interaction. As illustrated here, contacts between core transmembrane domains are likely; however, they have not been demonstrated experimentally. Conformational changes induced by binding of GABA result in the activation of the G_i/o G protein through specific interactions between Gα_i/o and the intracellular segments of GB2. GB1 intracellular segments may not be involved in the G protein coupling (A) or may provide contacts for the βγ subunit and/or conserved parts of the α subunit (B). Gray spheres represent GABA.