GABA (gamma-amino-butyric acid), the principal inhibitory neurotransmitter in the brain, signals through ionotropic (GABA(A)/ GABA(c)) and metabotropic (GABA(B)) receptor systems. Here we report the cloning of GABA(B) receptors. Photoaffinity labelling experiments suggest that the cloned receptors correspond to two highly conserved GABA(B) receptor forms present in the vertebrate nervous system. The cloned receptors negatively couple to adenylyl cyclase and show sequence similarity to the metabotropic receptors for the excitatory neurotransmitter L-glutamate.

The unit cell of poly-e-caprolactone was found to be orthorhombic with dimensions a = 7.496 + 0.002, b=4.974+0"001, c= 17.297+0-023/~ (fiber axis). The space group is P212121. This unit cell is only compatible with an extended planar chain conformation of the molecule involving two monomer residues related by a twofold screw axis in the chain direction. The P212~2~ space group and the density of 1.146 g.cm-3 indicate that the unit cell contains two chains with opposite orientation ('up' and 'down'). Intensity measurements and structure factor calculations require the rotation of the plane of the chains about their axis to an angle of 28 ° with respect to the a axis; longitudinal chain shift places the ester groups in planes perpendicular to the c axis. Folded chain single crystals with a lancet-like structure were observed by electron microscopy.

The ␥-aminobutyric acid type B (GABA B ) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABA B receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteinerich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABA B receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABA B receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABA B receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.

ABSTRACT␥-Aminobutyric acid type B receptors (GABA B Rs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABA B Rs inhibit neurotransmitter release by down-regulating high-voltage activated Ca 2؉ channels, whereas postsynaptic GABA B Rs decrease neuronal excitability by activating a prominent inwardly rectifying K ؉ (Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABA B Rs, hGABA B R1a (hR1a) and hGABA B R1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABA B Rs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1͞3.2 and Kir3.1͞3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABA B Rs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the GABA B R1 gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (EJM1) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.

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