Ca2+ Requirement for High-Affinity γ-Aminobutyric Acid (GABA) Binding at GABAB Receptors: Involvement of Serine 269 of the GABABR1 Subunit

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Abstract
The γ-aminobutyric acid (GABA) receptor type B (GABABR) is constituted of at least two homologous proteins, GABABR1 and GABABR2. 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 GABABR 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 GTPγS 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 μM. In cells expressing GABABR1, the potency of GABA, but not of baclofen, in displacing bound 125I-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 GABABR1 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 GABAB receptor function is no longer observed in cells coexpressing this GABABR1-S269A mutant and the wild-type GABABR2. Taken together, these results show that Ser269, which is conserved in the GABABR1 protein fromCaenorhabditis elegans to mammals, is critical for the Ca2+-effect on the heteromeric GABAB receptor.