Abstract

The γ-aminobutyric acid (GABA) receptor type B (GABA_BR) is constituted of at least two homologous proteins, GABA_BR1 and GABA_BR2. 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_BR 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 GABA_BR1, 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 GABA_BR1 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_BR1-S269A mutant and the wild-type GABA_BR2. Taken together, these results show that Ser269, which is conserved in the GABA_BR1 protein fromCaenorhabditis elegans to mammals, is critical for the Ca²⁺-effect on the heteromeric GABA_B receptor.