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

  1. Thierry Galvez1,
  2. Stephan Urwyler2,
  3. Laurent Prézeau1,
  4. Johannes Mosbacher2,
  5. Cécile Joly1,
  6. Barbara Malitschek2,
  7. Jakob Heid2,
  8. Isabelle Brabet1,
  9. Wolfgang Froestl2,
  10. Bernhard Bettler2,
  11. Klemens Kaupmann2 and
  12. Jean-Philippe Pin1
  1. 1Centre Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Pharmacologie-Endocrinologie, UPR 9023-Centre National de la Recherche Scientifique, Montpellier, France (T.G., L.P., C.J., I.B., J.-P.P.); and 2TA Nervous system, Novartis Pharma AG, Basel, Switzerland (S.U., J.M., B.M., J.H., W.F., B.B., K.K.).

    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.

    Footnotes

    • Send reprint requests to: Dr. Thierry Galvez, CCIPE-Centre National de la Recherche Scientifique-UPR9023, Laboratoire des Mécanismes Moléculaires des Communications Cellulaires, 141 rue de la Cardonille, F-34094 Montpellier Cedex 5, France. E-mail:galvez{at}ccipe.montp.inserm.fr

    • This work was supported by grants from the “Action Incitative Physique et Chimie du vivant” (PCV97–115), the European Community Biomed2 (BMH4-CT96–0228) and Biotech2 (BIO4-CT96–0049) programs, the Fondation pour la Recherche Médicale, Novartis Pharma (Basel, Switzerland) and the association Retina France (all to J.-P.P.)

    • Abbreviations:
      GABA
      γ-aminobutyric acid
      GPCR
      G protein-coupled receptor
      mGluR
      metabotropic glutamate receptor
      CaSR
      Ca2+-sensing receptor
      HEK
      human embryonic kidney
      GTPγS
      guanosine 5′-O-(3-thiotriphosphate)
      IP
      inositol phosphates
      APPA
      3-aminopropylphosphinic acid
      LBP
      leucine binding protein
      PLC
      phospholipase C
      LIVBP
      leucine/isoleucine/valine binding protein
      • Received July 30, 1999.
      • Accepted October 20, 1999.
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    1. Molecular Pharmacology March 1, 2000 vol. 57 no. 3 419-426
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