The ionotropic glutamate receptor GluR6 exhibits strongly and rapidly desensitizing current responses. Treatment of heterologically expressed GluR6 with the lectin concanavalin A (ConA) in Xenopus oocytes as well as in human embryonic kidney-293 cells results in a considerable increase of the steady-state current, presumably by inhibiting receptor desensitization. In the present study, we investigated the molecular basis of this effect using a systematic mutagenesis approach. We found that although N-glycosylation is an absolute prerequisite for the lectin-mediated inhibition of desensitization, no single one of the nine extracellular consensus sites for N-glycosylation of GluR6 is required. Rather, each of the nine N-linked carbohydrate side chains is independently capable of modulatory interaction with the lectin. Moreover, even artificially introduced N-glycosylation sites can substitute for native sites. Thus, the specific site of the lectin binding does not appear to be important for its desensitization-inhibiting action. Furthermore, we show that the extent of the receptor's ConA sensitivity depends on its state of activation, because the desensitized GluR6 exhibits significantly lower lectin sensitivity than the nondesensitized receptor. We conclude that binding of ConA "locks" the receptor in the activatable state, thereby inhibiting conformational changes required to shift the receptor to the desensitized state.

We have identified a splice variant encoding only the extracellular ligand-binding domain of the ␥-aminobutyric acid B (GABA B ) receptor subunit GABA B(1a) . This isoform, which we have named GABA B(1e) , is detected in both rats and humans. While GABA B(1e) is a minor component of the total pool of GABA B(1) transcripts detected in the central nervous system, it is the primary isoform found in all peripheral tissues examined. When expressed in a heterologous system, the truncated receptor is both secreted and membrane associated. However, GABA B(1e) lacks the ability to bind the radiolabeled antagonist [ 3 H]CGP 54626A, activate G-protein coupled inwardly rectifying potassium channels, or inhibit forskolin-induced cAMP production when it is expressed alone or together with GABA B(2) . Interestingly, when co-expressed with GABA B(2) , not only does the truncated receptor heterodimerize with GABA B(2) , the association is of sufficient avidity to disrupt the normal GABA B(1a) / GABA B(2) association. Despite this strong interaction, GABA B(1e) fails to disrupt G-protein coupled inwardly rectifying potassium activation by the full-length heterodimer pair of GABA B(1a) /GABA B(2) . ␥-Aminobutyric acid (GABA)1 is the primary neurotransmitter responsible for neuronal inhibition. The method by which inhibition is achieved depends upon the type, as well as the anatomical localization, of the GABA receptor. Like other neurotransmitters, inhibition is mediated through both ionotropic (GABA A ) and metabotropic (GABA B ) receptors. The ionotropic receptor subunits form ion channels that are selectively permeable to chloride. These receptors are responsible for the rapid component of inhibitory postsynaptic potentials. The metabotropic receptors are coupled to heterotrimeric G-proteins which in turn regulate intracellular effector systems. When located at a post-synaptic junction, these receptors manifest long lasting inhibitory postsynaptic potentials by increasing potassium conductance through G-protein coupled inwardly rectifying potassium channels (GIRKs). Those receptors located at a presynaptic junction control neurotransmitter release by inhibiting voltage-gated Ca 2ϩ channels. GABA B receptors belong to the class C subfamily of G-protein coupled receptors. These receptors, which include the calcium sensing (1), metabotropic glutamate (GRM) (2), vomeronasal (3), and putative taste receptors (4), share low sequence similarity. Like the classical G-protein Ca 2ϩ receptors, they are composed of seven-membrane spanning domains, with the intracellular loops being responsible for coupling to heterotrimeric G-proteins. However, they have the distinctive feature of an unusually large extracellular domain (ECD) that is nearly equal in size to the remaining portion of the protein, and bears similarity to the bacterial periplasmic amino acid-binding proteins (5, 6). The ECDs of GABA B(1a) , GRM1, and GRM4 have been successfully expressed as soluble recombinant proteins and possess ligand binding characteristics typical of th...

In this study, we describe the pharmacological characterization of novel aryl-ether, biaryl, and fluorene aspartic acid and diaminopropionic acid analogs as potent inhibitors of EAAT2, the predominant glutamate transporter in forebrain regions. The rank order of potency determined for the inhibition of human EAAT2 was(WAY-211686) (IC 50 ϭ 190 Ϯ 10 nM). WAY-213613 was the most selective of the compounds examined, with IC 50 values for inhibition of EAAT1 and EAAT3 of 5 and 3.8 M, respectively, corresponding to a 59-and 45-fold selectivity toward EAAT2. An identical rank order of potency [WAY-213613 (35 Ϯ 7 nM) Ͼ WAY-213394 (92 Ϯ 13 nM) ϭ WAY-212922 (95 Ϯ 8 nM) ϭ WAY-211686 (101 Ϯ 20 nM)] was observed for the inhibition of glutamate uptake in rat cortical synaptosomes, consistent with the predominant contribution of EAAT2 to this activity. Kinetic studies with each of the compounds in synaptosomes revealed a competitive mechanism of inhibition. All compounds were determined to be nonsubstrates by evaluating both the stimulation of currents in EAAT2-injected oocytes and the heteroexchange of D-[ 3 H]aspartate from cortical synaptosomes. WAY-213613 represents the most potent and selective inhibitor of EAAT2 identified to date. Taken in combination with its selectivity over ionotropic and metabotropic glutamate receptors, this compound represents a potential tool for the further elucidation of EAAT2 function.Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. Glutamate transmission is mediated via interaction with the ligand-gated ion channel receptors, termed the ionotropic receptors, and the seven-transmembrane domain G-protein-coupled receptors, termed metabotropic glutamate receptors (Barnard, 1997;Schoepp et al., 1999). Activation of these receptors is responsible for the physiological actions of glutamate, whereas paradoxically, overstimulation of the ionotropic receptors contributes to the excitotoxic actions attributed to glutamate. Therefore, synaptic glutamate levels must be tightly regulated to maintain the integrity of synaptic transmission and to limit or prevent the pathophysiological activity of this excitatory neurotransmitter.A family of high-affinity Na ϩ -dependent glutamate transporters expressed in the plasma membranes of both neurons and astroglia is responsible for the clearance of extracellular glutamate by mediating the cellular uptake of glutamate in a Article, publication date, and citation information can be found at

Peripheral corticotropin-releasing factor (CRF) receptor ligands inhibit gastric acid secretion and emptying while stimulating gastric mucosal blood flow in rats. Endogenous CRF ligands are expressed in the upper gastrointestinal (GI) tissues pointing to local expression of CRF receptors. We mapped the distribution of CRF receptor type 1 (CRF 1 ) and 2 (CRF 2 ) in the rat upper GI. Polyclonal antisera directed against the C-terminus of the CRF receptor protein were generated in rabbits and characterized by western blotting and immunofluorescence using CRF 1 -and CRF 2 -transfected cell lines and in primary cultured neurons from rat brain cortex. A selective anti-CRF 1 antiserum (4467a-CRF 1 ) was identified and used in parallel with another antiserum recognizing both CRF 1 and CRF 2 (4392a-CRF 1&2 ) to immunostain gastric tissue sections. Antiserum 4467a-CRF 1 demonstrated specific immunostaining in a narrow zone in the upper oxyntic gland within the stomach corpus.Conversely, 4392a-CRF 1&2 labeled cells throughout the oxyntic gland and submucosal blood vessels. Pre-absorption with the specific antigen peptide blocked immunostaining in all experiments. Doublestaining showed co-localization of 4392a-CRF 1&2 but not 4467a-CRF 1 immunoreactivity with H/ K-ATPase and somatostatin immunostaining in parietal and endocrine cells of the oxyntic gland. No specific staining was observed in the antrum with either antisera, whereas only antiserum 4392a-CRF 1&2 showed modest immunoreactivity in the duodenal mucosa. Finally, co-localization of CRF 2 and urocortin immunoreactivity was found in the gastric glands. These results indicate that both CRF receptor subtypes are expressed in the rat upper GI tissues with a distinct pattern and regional differences suggesting differential function.

Indiplon (NBI 34060) is a novel pyrazolopyrimidine currently in development for the treatment of insomnia. We have previously shown that indiplon exhibits high-affinity binding to native GABA A receptors from rat brain and acts as a positive allosteric modulator of GABA A receptor currents in cultured rat neurons (Sullivan et al., 2004). In this study, we examined the GABA A receptor ␣ subunit selectivity of indiplon using electrophysiological techniques to record GABA-activated chloride currents from recombinant rodent GABA A receptors expressed in human embryonic kidney 293 cells. Indiplon potentiated the GABA-activated chloride current in recombinant GABA A receptors in a dose-dependent and reversible manner and was approximately 10-fold selective for ␣1 subunit-containing receptors over GABA A receptors containing ␣2, ␣3, or ␣5 subunits. The EC 50 values were 2.6, 24, 60, and 77 nM for ␣1␤2␥2, ␣2␤2␥2, ␣3␤3␥2, and ␣5␤2␥2 receptors, respectively. Indiplon was approximately 10 times more potent than zolpidem and zopiclone and Ͼ100 times more potent than zaleplon. Moreover, indiplon, up to 1 M, did not potentiate GABA A receptors composed of ␣4␤2␥2 and ␣6␤2␥2 subunits. This mechanism of action is proposed to underlie the sedative-hypnotic effects of indiplon in animals and humans.GABA is the major inhibitory neurotransmitter in the mammalian central nervous system (Macdonald and Olsen, 1994). Drugs that enhance inhibitory neurotransmission include benzodiazepines, barbiturates, and general anesthetics and are used therapeutically as sedative-hypnotics, anxiolytics, antiepileptics, muscle relaxants, and anesthetics. These drugs target the GABA A receptor, an ion channel that selectively passes chloride when gated by the binding of GABA. Chloride influx serves to hyperpolarize or stabilize a negative resting membrane potential, making the neuron resistant to excitation.The GABA A receptor is a hetero-oligomeric complex composed of five transmembrane spanning subunits from sixteen different genes, ␣(1-6), ␤(1-3), ␥(1-3), ␦, ⑀, , and (Barnard et al., 1998;Korpi et al., 2002;Whiting, 2003). In most neurons, two ␣ subunits, two ␤ subunits, and one ␥ subunit form the typical GABA A receptor (Chang et al., 1996;Tretter et al., 1997). The ␦, ⑀, , and subunits have some reported selective functions but are not yet fully understood. Theoretically, there are thousands of possible subunit combinations, but a limited number of subtype combinations have been found in native systems with ␣1␤2␥2, ␣2␤3␥2, and ␣3␤3␥2 being the most abundant (Whiting, 2003). The assembly of ␣, ␤, and ␥ subunits is required to produce functional GABA A receptors that exhibit all of the pharmacological properties of native GABA A receptors. Benzodiazepine binding occurs at the interface between ␣ and ␥2 subunits (Wieland et al., 1992). GABA elicits chloride currents in recombinant GABA A receptors composed of only ␣ and ␤ subunits, but these currents are not potentiated by benzodiazepines (Schofield et al., 1987).The diversity of subunits and...

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4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-pyrimidines bearing substituted pyridyl groups as C-6 substituents were prepared as selective adenosine hA2A receptor antagonists for the treatment of Parkinson's disease. The 5-methoxy-3-pyridyl derivative 6g (hA2A Ki 2.3 nM, hA1 Ki 190 nM) was orally active at 3 mg/kg in a rat HIC model but exposure was poor in nonrodent species, presumably due to poor aqueous solubility. Follow-on compound 16a (hA2A Ki 0.83 nM, hA1 Ki 130 nM), bearing a 6-(morpholin-4-yl)-2-pyridyl substituent at C-6, had improved solubility and was orally efficacious (3 mg/kg, HIC) but showed time-dependent cytochrome P450 3A4 inhibition, possibly related to morpholine ring metabolism. Compound 16j (hA2A Ki 0.44 nM, hA1 Ki 80 nM), bearing a 6-(4-methoxypiperidin-1-yl)-2-pyridyl substituent at C-6, was sparingly soluble but had good oral exposure in rodent and nonrodent species, had no cytochrome P450 or human ether-a-go-go related gene channel issues, and was orally efficacious at 1 mg/kg in HIC and at 3 mg/kg for potentiation of l-dopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats.

A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.