B-type receptors for the neurotransmitter GABA (gamma-aminobutyric acid) inhibit neuronal activity through G-protein-coupled second-messenger systems, which regulate the release of neurotransmitters and the activity of ion channels and adenylyl cyclase. Physiological and biochemical studies show that there are differences in drug efficiencies at different GABA(B) receptors, so it is expected that GABA(B)-receptor (GABA(B)R) subtypes exist. Two GABA(B)-receptor splice variants have been cloned (GABA(B)R1a and GABA(B)R1b), but native GABA(B) receptors and recombinant receptors showed unexplained differences in agonist-binding potencies. Moreover, the activation of presumed effector ion channels in heterologous cells expressing the recombinant receptors proved difficult. Here we describe a new GABA(B) receptor subtype, GABA(B)R2, which does not bind available GABA(B) antagonists with measurable potency. GABA(B)R1a, GABA(B)R1b and GABA(B)R2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. We provide evidence for the assembly of heteromeric GABA(B) receptors in vivo and show that GABA(B)R2 and GABA(B)R1a/b proteins immunoprecipitate and localize together at dendritic spines. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial-agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABA(B) receptor. Heteromeric assembly among G-protein-coupled receptors has not, to our knowledge, been described before.

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

Assembly of fully functional GABABreceptors requires heteromerization of the GABAB(1)and GABAB(2)subunits. It is thought that GABAB(1)and GABAB(2)undergo coiled-coil dimerization in their cytoplasmic C termini and that assembly is necessary to overcome GABAB(1)retention in the endoplasmatic reticulum (ER). We investigated the mechanism underlying GABAB(1)trafficking to the cell surface. We identified a signal, RSRR, proximal to the coiled-coil domain of GABAB(1)that when deleted or mutagenized allows for surface delivery in the absence of GABAB(2). A similar motif, RXR, was recently shown to function as an ER retention/retrieval (ERR/R) signal in KATPchannels, demonstrating that G-protein-coupled receptors (GPCRs) and ion channels use common mechanisms to control surface trafficking. A C-terminal fragment of GABAB(2)is able to mask the RSRR signal and to direct the GABAB(1)monomer to the cell surface, where it is functionally inert. This indicates that in the heteromer, GABAB(2)participates in coupling to the G-protein. Mutagenesis of the C-terminal coiled-coil domains in GABAB(1)and GABAB(2)supports the possibility that their interaction is involved in shielding the ERR/R signal. However, assembly of heteromeric GABABreceptors is possible in the absence of the C-terminal domains, indicating that coiled-coil interaction is not necessary for function. Rather than guaranteeing heterodimerization, as previously assumed, the coiled-coil structure appears to be important for export of the receptor complex from the secretory apparatus.

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.

Summary: Conventional approaches to produce transgenic mice recurrently yield unpredictable patterns and levels of transgene expression, a situation calling for the development of new techniques to overcome these drawbacks in the context of overexpression studies. Here we present an efficient method for rapid and reproducible transgenesis using the recombinase mediated cassette exchange (RMCE) (Bouhassira et al.: Blood 90:3332-3344, 1997) procedure. A lox511-EGFP-TK/neoloxP cassette was placed under the control of the endogenous mouse b-actin promoter. Heterozygous mice revealed strong and ubiquitous EGFP expression throughout embryogenesis and adulthood. Reproducibly, the same expression pattern was obtained with RMCE when it was used to replace the EGFP-harboring cassette by ECFP or placental alkaline phosphatase (PLAP) reporter genes (DePrimo et al.: Transgenic Res 5:459-466, 1996). Furthermore, the RMCE procedure proved efficient as well in embryonic stem (ES) Key words: AGRP; targeted transgenesis; RMCE; mouse; lox511As a consequence of unpredictable positional effects and varying copy numbers, transgenic mice, generated by conventional DNA injection techniques, commonly present incoherent transgene expression patterns. Therefore, it is often necessary to analyze multiple mouse lines before conclusions, as to the effect of overexpressed transgenic products, can be drawn. In contrast, targeting transgenes into defined and precharacterized loci yields predictable expression patterns due to the invariable transcriptional control exerted by the given endogenous regulatory sequences.In order to reproducibly obtain strong and ubiquitous transgene expression in mice, we have therefore focused investigations on endogenous b-actin, a locus consistently expressed during embryonic development and adulthood. The b-actin genomic region was cloned by screening a BAC library with intron 3 and used to generate the targeting vector by inserting a lox511-EGFPTKNeo-loxP cassette at the b-actin translation start codon in exon 2 (Fig. 1a). Strong EGFP expression was observed in the targeted BALB/c ES-cell clones, as well as in mice which were generated from these ( Fig. 2). Mice heterozygous for the targeted b-actin-EGFP allele were fertile and presented strong, ubiquitous EGFP fluorescence throughout embryogenesis (starting at embryonic day (E)2.5) (Fig. 2b) and in adult organs such as brain, eye, heart, liver, lung, kidney, muscle, thymus, spleen, small intestine, stomach, large intestine, skin, uterus, and tail (Fig. 2a). Except for a 10% lower body weight, X-ray analysis, blood chemo-gram, hemogram, urine analysis, and histopathology all failed to reveal differences with respect to wildtype animals. Homozygous mice, however, were not viable and died between days E10.5 and E11.5 (Fig. 2b) (and as published previously;Shawlot et al., 1998).The b-actin-EGFP ES cells were used to test the efficiency of Cre-recombinase-mediated cassette exchange (RMCE) of lox511-loxP flanked transgenes.Heterospecific lox-sites (wildtype loxP...