Ca2+ requirement for high-affinity gamma-aminobutyric acid (GABA) binding at GABA(B) receptors: Involvement of serine 269 of the GABA(B)R1 subunit

The gamma-aminobutyric acid (GABA) receptor type B (GABA(B)R) is constitute d of at least two homologous proteins, GABA(B)R1 and GABA(B)R2. These prote ins share sequence and structural similarity with metabotropic glutamate an d Ca2+-sensing receptors, both of which are sensitive to Ca2+. Using rat br ain membranes, we report here that the affinity of GABA and 3-aminopropylph osphinic 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 po tency 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 r egulating GABA affinity was 37 mu M. In cells expressing GABA(B)R1, the pot ency of GABA, but not of baclofen, in displacing bound I-125-CGP64213 was s imilarly decreased in the absence of Ca2+. To identify residues that are re sponsible for the Ca2+ effect, the pharmacological profile and the Ca2+ sen sitivity of a series of GABA(B)R1 mutants were examined. The mutation of Se r269 into Ala was found to decrease the affinity of GABA, but not of baclof en, and the GABA affinity was found not to be affected upon Ca2+ removal. F inally, the effect of Ca2+ on the GABA(B) receptor function is no longer ob served 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 conserv ed in the GABA(B)R1 protein from Caenorhabditis elegans to mammals, is crit ical for the Ca2+-effect on the heteromeric GABA(B) receptor.