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

The g-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 Ca-sensing receptors, both of which are sensitive to Ca. 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 Ca. Such a large effect of Ca is not observed with baclofen or the antagonists CGP64213 and CGP56999A. In contrast to baclofen, the potency of GABA in stimulating GTPgS binding in rat brain membranes is also decreased by a factor .10 upon Ca removal. The potency for Ca in regulating GABA affinity was 37 mM. In cells expressing GABABR1, the potency of GABA, but not of baclofen, in displacing bound I-CGP64213 was similarly decreased in the absence of Ca. To identify residues that are responsible for the Ca effect, the pharmacological profile and the Ca 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 Ca removal. Finally, the effect of Ca 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 from Caenorhabditis elegans to mammals, is critical for the Ca-effect on the heteromeric GABAB receptor. The neurotransmitter g-aminobutyric acid (GABA) activates both ionotropic receptors (the GABAA and GABAC receptors) and G protein-coupled metabotropic receptors (the GABAB receptors). GABAA and GABAC receptors are permeable to Cl ions and mediate, in most cases, fast inhibition of the postsynaptic neurons. GABAB receptors mostly couple to pertussis toxin-sensitive G proteins (Gi or Go) and accordingly inhibit the activity of adenylyl cyclase and voltagegated Ca-channels or activate G protein-regulated inwardly rectifying K-channels (Kir3) (for reviews, see Kerr and Ong, 1995; Bettler et al., 1998). When located on the postsynaptic element, the GABAB receptors inhibit neuronal excitability (Kerr and Ong, 1995; Bettler et al., 1998). GABAB receptors are also found on glutamatergic or GABA-ergic terminals, where they inhibit neurotransmitter release. GABAB receptors are widely expressed in the central nervous system, both in brain and spinal cord. Accordingly, these receptors play important physiological roles in pain transmission, absence epilepsy, and drug addiction (Kerr and Ong, 1995; Bettler et al., 1998). Two GABAB receptor proteins have been identified: GABABR1 (Kaupmann et al., 1997) and GABABR2 (Jones et al., 1998; Kaupmann et al., 1998b; White et al., 1998; Kuner et al., 1999; Ng et al., 1999). Neither receptor was found to efficiently couple to G proteins when expressed alone (Kaupmann et al., 1997, 1998a,b; Martin et al., 1999). Indeed, GABAB receptors that efficiently activate pertussis-toxinsensitive G proteins were only obtained in cells coexpressing GABABR1 and GABABR2 (Jones et al., 1998; Kaupmann et al., 1998b; White et al., 1998; Kuner et al., 1999; Ng et al., This work was supported by grants from the “Action Incitative Physique et Chimie du vivant” (PCV97–115), the European Community Biomed2 (BMH4CT96–0228) 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, g-aminobutyric acid; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; CaSR, Casensing receptor; HEK, human embryonic kidney; GTPgS, guanosine 59-O-(3-thiotriphosphate); IP, inositol phosphates; APPA, 3-aminopropylphosphinic acid; LBP, leucine binding protein; PLC, phospholipase C; LIVBP, leucine/isoleucine/valine binding protein. 0026-895X/00/030419-08$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 57:419–426 (2000). 419 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from 1999). Moreover, these two receptors were found to associate at least by their carboxyl-terminal sequence (White et al., 1998; Kuner et al., 1999), further suggesting that GABAB receptors function in a heteromeric form. Both GABABR1 and R2 proteins share sequence similarity with family 3 G protein-coupled receptors (GPCRs) (Bockaert and Pin, 1999), which include metabotropic glutamate receptors (mGluRs), Ca-sensing receptors (CaSR) and putative pheromone and taste receptors. Like all family 3 GPCRs, GABAB receptors possess a large amino-terminal domain structurally related to some bacterial periplasmic proteins, where ligands bind (Galvez et al., 1999; Malitschek et al., 1999). This aminoterminal domain is attached to a heptahelical transmembrane domain that constitutes the G protein-activating domain, as demonstrated for mGluRs (Pin et al., 1994; Gomeza