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With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGAT(flox/flox) mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT(-/-) mice were the same as Cx57-VGAT(+/+) controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT(-/-) mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT(-/-) mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors.

The absence of a2* nicotinic acetylcholine receptors (nAChRs) in oriens lacunosum moleculare (OLM) GABAergic interneurons ablate the facilitation of nicotine-induced hippocampal CA1 long-term potentiation and impair memory. The current study delineated whether genetic mutations of a2* nAChRs ("Chrna2"[superscript L9'S/L9'S] and "Chrna2"[superscript KO]) influence hippocampus-dependent learning and memory and CA1 synaptic plasticity. We substituted a serine for a leucine (L9'S) in the a2 subunit (encoded by the "Chrna2" gene) to make a hypersensitive nAChR. Using a dorsal hippocampus-dependent task of preexposure-dependent contextual fear conditioning, adolescent hypersensitive "Chrna2" [superscript L9'S/L9'S] male mice exhibited impaired learning and memory. The deficit was rescued by low-dose nicotine exposure. Electrophysiological studies demonstrated that hypersensitive a2 nAChRs potentiate acetylcholine-induced ion channel flux in oocytes and acute nicotine-induced facilitation of dorsal/intermediate CA1 hippocampal long-term potentiation in "Chrna2"[superscript L9'S/L9'S] mice. Adolescent male mice null for the a2 nAChR subunit exhibited a baseline deficit in learning that was not reversed by an acute dose of nicotine. These effects were not influenced by locomotor, sensory or anxiety-related measures. Our results demonstrated that a2* nAChRs influenced hippocampus-dependent learning and memory, as well as nicotine-facilitated CA1 hippocampal synaptic plasticity.

Although homomeric channels assembled from the [alpha]9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both [alpha]9 and [alpha]10 subunits. To gain insight into [alpha]10 subunit function in vivo, we examined olivocochlear innervation and function in [alpha]10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8-9 inner hair cells revealed ACh-gated currents in [alpha][10.sup.+/+] and [alpha][10.sup.+/-] mice, with no detectable responses to ACh in [alpha][10.sup.-/-] mice. In contrast, a proportion of [alpha][10.sup.-/-] outer hair cells showed small ACh-evoked currents. In [alpha][10.sup.-/-] mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual [alpha]9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, [alpha][10.sup.-/-] mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that [alpha][9.sup.-/-] and [alpha][10.sup.-/-] mice have overlapping but nonidentical phenotypes. Moreover, [alpha]10 nAChR subunits are required for normal olivocochlear activity because [alpha]9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in [alpha][10.sup.-/-] mutant mice. cochlea | electrophysiology | inner hair cells | outer hair cells

DSL (Delta, Serrate, Lag-2) ligands activate Notch signaling and thereby regulate the differentiation of many different cell types during development. We have isolated a novel Serrate-like gene, Jagged2, whose amino acid sequence and expression pattern during rat embryogenesis suggest that it functions as a ligand for Notch. In contrast to previously described DSL Ligands for Notch, Jagged2 is not widely expressed in the developing central nervous system. However, Jagged2 and Notch1 are coexpressed in the apical ectodermal ridge (AER), suggesting a role for this ligand-receptor pair in limb development. Furthermore, unlike Jagged1, Jagged2 is coexpressed with Notch in the developing thymus, where it may induce Notch signaling to direct T-cell fate. Our data are consistent with the idea that the diversity of cell types regulated by Notch signaling is a consequence of activation of unique Notch isoforms by different DSL ligands.

Cell culture assay studies find that Jagged activates rat Notch1 expressed in myoblasts and prevents muscle cell differentiation. Jagged, a rat cDNA clone, encodes a ligand for the vertebrate Notch protein. Some invertebrate ligands for the LIN 12/Notch have amino acid sequences similar to those ofJagged. Gene expression patterns for Jagged overlap with those of Notch1, Notch2 and Notch3.

In situ hybridization analyses of the alpha9 acetylcholine receptor isolated from rat cochlear hair cells reveal that alpha9 receptor has nicotinic-muscarinic pharmacological properties that resemble those of the cholinergic receptor in hair cells. Alpha9 gene expression is detectable in the outer cochlear hair cells, indicating a limited expression pattern. The alpha9 receptor mediates signal transduction between cochlear hair cells and efferent neuronal terminals and cholinergic efferent innervation.

We report the cloning and characterization of rat [Alpha]10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the [Alpha]10 nAChR subunit gene is most similar to the rat [Alpha]9 nAChR, and both [Alpha]9 and [Alpha]10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with [Alpha]10 cRNA alone or in pairwise combinations with either [Alpha]2-[Alpha]6 or [Beta]2-[Beta]4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of [Alpha]9 and [Alpha]10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric [Alpha]9 channels, the [Alpha]9[Alpha]10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct currentvoltage relationship, and a biphasic response to changes in extracellular [Ca.sup.2+] ions. The pharmacological profiles of homomeric [Alpha]9 and heteromeric [Alpha]9[Alpha]10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both [Alpha]9 and [Alpha]10 subunits.

Knock-in mice were generated that harbored a leucine-to-serine mutation in the [Alpha]4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.

Functional Expression of a New Pharmacological Subtype of Brain Nicotinic Acetycholine Receptor THERE IS A FAMILY OF GENES THAT encodes functional subunits of rat neuronal nicotinic acetylcholine receptors (nAChRs) (1-3). [...]

Baseline and nicotine-modulated behaviors were assessed in mice harboring a null mutant allele of the nicotinic acetylcholine receptor (nAChR) subunit gene α2 (Chrna2). Homozygous Chrnal mice are viable, show expected sex and Mendelian genotype ratios, and exhibit no gross neuroanatomical abnormalities. A broad range of behavioral tests designed to assess genotype-dependent effects on anxiety (elevated plus maze and light/dark box), motor coordination (narrow bean traverse and gait), and locomotor activity revealed no significant differences between mutant mice and age-matched wild-type littermates. Furthermore, a panel of tests measuring traits, such as body position, spontaneous activity, respiration, tremors, body tone, and startle response, revealed normal responses for Chrnal-mill mutant mice. However, Chrna2 -/- mice do exhibit a mild motor or coordination phenotype (a decreased latency to fall during the accelerating rotarod test) and possess an increased sensitivity to nicotine-induced analgesia in the hotplate assay. Relative to wild-type, Chrna2-/- mice show potentiated nicotine self-administration and withdrawal behaviors and exhibit a sex-dependent enhancement of nicotine-facilitated cued, but not trace or contextual, fear conditioning. Overall, our results suggest that loss of the mouse nAChR α2 subunit has very limited effects on baseline behavior but does lead to the potentiation of several nicotine-modulated behaviors. [ABSTRACT FROM AUTHOR]