ABSTRACT
The rodent OR-I7 is an olfactory receptor exemplar activated by aliphatic aldehydes such as octanal. Normal alkanals shorter than heptanal bind OR-I7 without activating it and hence function as antagonists in vitro. We report a series of aldehydes designed to probe the structural requirements for aliphatic ligand chains too short to meet the minimum approximate 6.9 Å length requirement for receptor activation. Experiments using recombinant mouse OR-I7 expressed in heterologous cells show that in the context of short aldehyde antagonists, OR-I7 prefers binding aliphatic chains without branches, though a single methyl on carbon-3 is permitted. The receptor can accommodate a surprisingly large number of carbons (e.g. ten in adamantyl) as long as the carbons are part of a conformationally constrained ring system. A rhodopsin-based homology model of mouse OR-I7 docked with the new antagonists suggests that small alkyl branches on the alkyl chain sterically interfere with the hydrophobic residues lining the binding site, but branch carbons can be accommodated when tied back into a compact ring system like the adamantyl and bicyclo[2.2.2]octyl systems.
Subject(s)
Aldehydes/chemistry , Receptors, Odorant/chemistry , Animals , Binding Sites , Ligands , Mice , Molecular Docking Simulation , Molecular Structure , Receptors, Odorant/antagonists & inhibitorsABSTRACT
Ketamine elicits various neuropharmacological effects, including sedation, analgesia, general anesthesia, and antidepressant activity. Through an in vitro screen, we identified four mouse olfactory receptors (ORs) that responded to ketamine. In addition to their presence in the olfactory epithelium, these G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) are distributed throughout the central nervous system. To better understand the molecular basis of the interactions between ketamine and ORs, we used sequence comparison and molecular modeling to design mutations that (i) increased, reduced, or abolished ketamine responsiveness in responding receptors, and (ii) rendered nonresponding receptors responsive to ketamine. We showed that olfactory sensory neurons (OSNs) that expressed distinct ORs responded to ketamine in vivo, suggesting that ORs may serve as functional targets for ketamine. The ability to both abolish and introduce responsiveness to ketamine in GPCRs enabled us to identify and confirm distinct interaction loci in the binding site, which suggested a signature ketamine-binding pocket that may guide exploration of additional receptors for this general anesthetic drug.
Subject(s)
Anesthetics, Dissociative/metabolism , Brain/metabolism , Ketamine/metabolism , Models, Molecular , Receptors, Odorant/genetics , Receptors, Odorant/metabolism , Animals , Base Sequence , Binding Sites/genetics , Luciferases , Mice , Molecular Dynamics Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Conformation , Receptors, Odorant/chemistry , Sequence Analysis, DNAABSTRACT
The mammalian odorant receptors (ORs) form a chemical-detecting interface between the atmosphere and the nervous system. This large gene family is composed of hundreds of membrane proteins predicted to form as many unique small molecule binding niches within their G-protein coupled receptor (GPCR) framework, but very little is known about the molecular recognition strategies they use to bind and discriminate between small molecule odorants. Using rationally designed synthetic analogs of a typical aliphatic aldehyde, we report evidence that among the ORs showing specificity for the aldehyde functional group, a significant percentage detect the aldehyde through its ability to react with water to form a 1,1-geminal (gem)-diol. Evidence is presented indicating that the rat OR-I7, an often-studied and modeled OR known to require the aldehyde function of octanal for activation, is likely one of the gem-diol activated receptors. A homology model based on an activated GPCR X-ray structure provides a structural hypothesis for activation of OR-I7 by the gem-diol of octanal.
Subject(s)
Aldehydes/metabolism , Receptors, Odorant/metabolism , Animals , RatsABSTRACT
Angelman syndrome (AS) is a severe disorder of postnatal brain development caused by neuron-specific loss of the HECT (homologous to E6AP carboxy terminus) domain E3 ubiquitin ligase Ube3a/E6AP. The cellular role of Ube3a remains enigmatic despite recent descriptions of synaptic and behavioral deficits in AS mouse models. Although neuron-specific imprinting is thought to limit the disease to the brain, Ube3a is expressed ubiquitously, suggesting a broader role in cellular function. In the current study, we demonstrate a profound structural disruption and cisternal swelling of the Golgi apparatus (GA) in the cortex of AS (UBE3A(m-/p+)) mice. In Ube3a knockdown cell lines and UBE3A(m-/p+) cortical neurons, the GA is severely under-acidified, leading to osmotic swelling. Both in vitro and in vivo, the loss of Ube3a and corresponding elevated pH of the GA is associated with a marked reduction in protein sialylation, a process highly dependent on intralumenal Golgi pH. Altered ion homeostasis of the GA may provide a common cellular pathophysiology underlying the diverse plasticity and neurodevelopmental deficits associated with AS.
Subject(s)
Cerebral Cortex/ultrastructure , Golgi Apparatus/genetics , Golgi Apparatus/pathology , N-Acetylneuraminic Acid/metabolism , Ubiquitin-Protein Ligases/metabolism , Analysis of Variance , Angelman Syndrome/genetics , Angelman Syndrome/pathology , Animals , Animals, Newborn , Bacterial Proteins/genetics , Cells, Cultured , Cerebral Cortex/cytology , Cytoplasmic Structures/genetics , Cytoplasmic Structures/metabolism , Cytoplasmic Structures/ultrastructure , Disease Models, Animal , Embryo, Mammalian , Female , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Glycine/analogs & derivatives , Golgi Apparatus/ultrastructure , Green Fluorescent Proteins/genetics , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Lectins/metabolism , Luminescent Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Mutagenesis , Neurons/metabolism , Neurons/ultrastructure , Protein Transport/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Spermine/analogs & derivatives , Transfection , Ubiquitin-Protein Ligases/deficiency , Vesicle-Associated Membrane Protein 2/genetics , Vesicle-Associated Membrane Protein 2/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Fluorescence recovery after photobleaching has been widely used to study dynamic processes in the cell, but less frequently to analyze binding interactions and extract binding constants. Here we use it to analyze gamma-tubulin binding to the mitotic spindle and centrosomes to determine the role of gamma-tubulin in microtubule nucleation in the spindle. We find rapid gamma-tubulin turnover in mitotic spindles of Drosophila early embryos, characterized by diffusional interactions and weak binding, differing from centrosomes with tight binding interactions. The diffusion coefficient of gamma-tubulin is consistent with a major species existing in the cytoplasm as the less efficiently nucleating gamma-tubulin small complex (gammaTuSC) or gamma-tubulin, rather than gamma-tubulin ring complex (gammaTuRC). The fluorescence recovery kinetics we observe implies that gamma-tubulin functions by binding weakly to spindle microtubules. gamma-Tubulin may interact transiently with the spindle, nucleating microtubules very rapidly, differing from centrosomes, where gamma-tubulin binds tightly to nucleate microtubules.
