Ca2+ requirement for high-affinity γ-aminobutyric acid (GABA) binding at GABAB receptors:: Involvement of serine 269 of the GABABR1 subunit

The gamma-aminobutyric acid (GABA) receptor type B (GABA(B)R) is constituted of at least two homologous proteins, GABA(B)R1 and GABA(B)R2. These proteins share sequence and structural similarity with metabotropic glutamate and Ca2+-sensing receptors, both of which are sensitive to Ca2+. Using rat brain membranes, we report here that the affinity of GABA and 3-aminopropylphosphinic acid for the GABA(B)R receptor is decreased by a factor >10 in the absence of Ca2+. Such a large effect of Ca2+ is not observed with baclofen or the antagonists CGP64213 and CGP56999A. In contrast to baclofen, the potency of GABA in stimulating GTP gamma S binding in rat brain membranes is also decreased by a factor >10 upon Ca2+ removal. The potency for Ca2+ in regulating GABA affinity was 37 mu M. In cells expressing GABA(B)R1, the potency of GABA, but not of baclofen, in displacing bound I-125-CGP64213 was similarly decreased in the absence of Ca2+. To identify residues that are responsible for the Ca2+ effect, the pharmacological profile and the Ca2+ sensitivity of a series of GABA(B)R1 mutants were examined. The mutation of Ser269 into Ala was found to decrease the affinity of GABA, but not of baclofen, and the GABA affinity was found not to be affected upon Ca2+ removal. Finally, the effect of Ca2+ on the GABA(B) receptor function is no longer observed in cells coexpressing this GABA(B)R1-S269A mutant and the wild-type GABA(B)R2. Taken together, these results show that Ser269, which is conserved in the GABA(B)R1 protein from Caenorhabditis elegans to mammals, is critical for the Ca2+-effect on the heteromeric GABA(B) receptor.