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

• Published in: Molecular pharmacology Vol. 57; no. 3; p. 419
• Main Authors: Thierry Galvez, Stephan Urwyler, Laurent Prézeau, Johannes Mosbacher, Cécile Joly, Barbara Malitschek, Jakob Heid, Isabelle Brabet, Wolfgang Froestl, Bernhard Bettler, Klemens Kaupmann, Jean-Philippe Pin
• Format: Journal Article
• Language: English
• Published: American Society for Pharmacology and Experimental Therapeutics 01.03.2000
• ISSN: 0026-895X; 1521-0111

The γ-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 Ca 2+ -sensing receptors, both of which are sensitive to Ca 2+ . 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 Ca 2+ . Such a large effect of Ca 2+ 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 Ca 2+ removal. The potency for Ca 2+ in regulating GABA affinity was 37 μM. In cells expressing GABA B R1, the potency of GABA, but not of baclofen, in displacing bound 125 I-CGP64213 was similarly decreased in the absence of Ca 2+ . To identify residues that are responsible for the Ca 2+ effect, the pharmacological profile and the Ca 2+ 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 Ca 2+ removal. Finally, the effect of Ca 2+ 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 Ca 2+ -effect on the heteromeric GABA B receptor.