Publications by authors named "Charles W Luetje"

51 Publications

Trading amino acids at the aphid- symbiotic interface.

Proc Natl Acad Sci U S A 2019 08 23;116(32):16003-16011. Epub 2019 Jul 23.

Institute for Cell & Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, NE2 4HH, Newcastle upon Tyne, United Kingdom;

Plant sap-feeding insects are widespread, having evolved to occupy diverse environmental niches despite exclusive feeding on an impoverished diet lacking in essential amino acids and vitamins. Success depends exquisitely on their symbiotic relationships with microbial symbionts housed within specialized eukaryotic bacteriocyte cells. Each bacteriocyte is packed with symbionts that are individually surrounded by a host-derived symbiosomal membrane representing the absolute host-symbiont interface. The symbiosomal membrane must be a dynamic and selectively permeable structure to enable bidirectional and differential movement of essential nutrients, metabolites, and biosynthetic intermediates, vital for growth and survival of host and symbiont. However, despite this crucial role, the molecular basis of membrane transport across the symbiosomal membrane remains unresolved in all bacteriocyte-containing insects. A transport protein was immunolocalized to the symbiosomal membrane separating the pea aphid from its intracellular symbiont The transporter, nonessential amino acid transporter 1, or ApNEAAT1 (gene: ), was characterized functionally following heterologous expression in oocytes, and mediates both inward and outward transport of small dipolar amino acids (serine, proline, cysteine, alanine, glycine). Electroneutral ApNEAAT1 transport is driven by amino acid concentration gradients and is not coupled to transmembrane ion gradients. Previous metabolite profiling of hemolymph and bacteriocyte, alongside metabolic pathway analysis in host and symbiont, enable prediction of a physiological role for ApNEAAT1 in bidirectional host-symbiont amino acid transfer, supplying both host and symbiont with indispensable nutrients and biosynthetic precursors to facilitate metabolic complementarity.
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http://dx.doi.org/10.1073/pnas.1906223116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690024PMC
August 2019

Use of machine learning to identify novel, behaviorally active antagonists of the insect odorant receptor co-receptor (Orco) subunit.

Sci Rep 2019 03 11;9(1):4055. Epub 2019 Mar 11.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, 33136, USA.

Olfaction is a key component of the multimodal approach used by mosquitoes to target and feed on humans, spreading various diseases. Current repellents have drawbacks, necessitating development of more effective agents. In addition to variable odorant specificity subunits, all insect odorant receptors (ORs) contain a conserved odorant receptor co-receptor (Orco) subunit which is an attractive target for repellent development. Orco directed antagonists allosterically inhibit odorant activation of ORs and we previously showed that an airborne Orco antagonist could inhibit insect olfactory behavior. Here, we identify novel, volatile Orco antagonists. We functionally screened 83 structurally diverse compounds against Orco from Anopheles gambiae. Results were used for training machine learning models to rank probable activity of a library of 1280 odorant molecules. Functional testing of a representative subset of predicted active compounds revealed enrichment for Orco antagonists, many structurally distinct from previously known Orco antagonists. Novel Orco antagonist 2-tert-butyl-6-methylphenol (BMP) inhibited odorant responses in electroantennogram and single sensillum recordings in adult Drosophila melanogaster and inhibited OR-mediated olfactory behavior in D. melanogaster larvae. Structure-activity analysis of BMP analogs identified compounds with improved potency. Our results provide a new approach to the discovery of behaviorally active Orco antagonists for eventual use as insect repellents/confusants.
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http://dx.doi.org/10.1038/s41598-019-40640-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411751PMC
March 2019

Mutant cycle analysis identifies a ligand interaction site in an odorant receptor of the malaria vector .

J Biol Chem 2017 11 29;292(46):18916-18923. Epub 2017 Sep 29.

From the Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33101

Lack of information about the structure of insect odorant receptors (ORs) hinders the development of more effective repellants to control disease-transmitting insects. Mutagenesis and functional analyses using agonists to map the odorant-binding sites of these receptors have been limited because mutations distant from an agonist-binding site can alter agonist sensitivity. Here we use mutant cycle analysis, an approach for exploring the energetics of protein-protein or protein-ligand interactions, with inhibitors, to identify a component of the odorant-binding site of an OR from the malaria vector, The closely related odorant-specificity subunits Agam/Or15 and Agam/Or13 were each co-expressed with Agam/Orco (odorant receptor co-receptor subunit) in oocytes and assayed by two-electrode voltage clamp electrophysiology. We identified (-)-fenchone as a competitive inhibitor with different potencies at the two receptors and used this difference to screen a panel of 37 Agam/Or15 mutants, surveying all positions that differ between Agam/Or15 and Agam/Or13 in the transmembrane and extracellular regions, identifying position 195 as a determinant of (-)-fenchone sensitivity. Inhibition by (-)-fenchone and six structurally related inhibitors of Agam/Or15 receptors containing each of four different hydrophobic residues at position 195 served as input data for mutant cycle analysis. Several mutant cycles, calculated from the inhibition of two receptors by each of two ligands, yielded coupling energies of ≥1 kcal/mol, indicating a close, physical interaction between the ligand and residue 195 of Agam/Or15. This approach should be useful in further expanding our knowledge of odorant-binding site structures in ORs of disease vector insects.
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http://dx.doi.org/10.1074/jbc.M117.810374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704475PMC
November 2017

Mammalian odorant receptor tuning breadth persists across distinct odorant panels.

PLoS One 2017 25;12(9):e0185329. Epub 2017 Sep 25.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

The molecular receptive range (MRR) of a mammalian odorant receptor (OR) is the set of odorant structures that activate the OR, while the distribution of these odorant structures across odor space is the tuning breadth of the OR. Variation in tuning breadth is thought to be an important property of ORs, with the MRRs of these receptors varying from narrowly to broadly tuned. However, defining the tuning breadth of an OR is a technical challenge. For practical reasons, a screening panel that broadly covers odor space must be limited to sparse coverage of the many potential structures in that space. When screened with such a panel, ORs with different odorant specificities, but equal tuning breadths, might appear to have different tuning breadths due to chance. We hypothesized that ORs would maintain their tuning breadths across distinct odorant panels. We constructed a new screening panel that was broadly distributed across an estimated odor space and contained compounds distinct from previous panels. We used this new screening panel to test several murine ORs that were previously characterized as having different tuning breadths. ORs were expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp electrophysiology. MOR256-17, an OR previously characterized as broadly tuned, responded to nine novel compounds from our new screening panel that were structurally diverse and broadly dispersed across an estimated odor space. MOR256-22, an OR previously characterized as narrowly tuned, responded to a single novel compound that was structurally similar to a previously known ligand for this receptor. MOR174-9, a well-characterized receptor with a narrowly tuned MRR, did not respond to any novel compounds in our new panel. These results support the idea that variation in tuning breadth among these three ORs is not an artifact of the screening protocol, but is an intrinsic property of the receptors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185329PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612731PMC
October 2017

Inhibition of insect olfactory behavior by an airborne antagonist of the insect odorant receptor co-receptor subunit.

PLoS One 2017 31;12(5):e0177454. Epub 2017 May 31.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

Response to volatile environmental chemosensory cues is essential for insect survival. The odorant receptor (OR) family is an important class of receptors that detects volatile molecules; guiding insects towards food, mates, and oviposition sites. ORs are odorant-gated ion channels, consisting of a variable odorant specificity subunit and a conserved odorant receptor co-receptor (Orco) subunit, in an unknown stoichiometry. The Orco subunit possesses an allosteric site to which modulators can bind and noncompetitively inhibit odorant activation of ORs. In this study, we characterized several halogen-substituted versions of a phenylthiophenecarboxamide Orco antagonist structure. Orco antagonist activity was assessed on ORs from Drosophila melanogaster flies and Culex quinquefasciatus mosquitoes, expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp electrophysiology. One compound, OX1w, was also shown to inhibit odorant activation of a panel of Anopheles gambiae mosquito ORs activated by diverse odorants. Next, we asked whether Orco antagonist OX1w could affect insect olfactory behavior. A Drosophila melanogaster larval chemotaxis assay was utilized to address this question. Larvae were robustly attracted to highly diluted ethyl acetate in a closed experimental chamber. Attraction to ethyl acetate was Orco dependent and also required the odorant specificity subunit Or42b. The addition of the airborne Orco antagonist OX1w to the experimental chamber abolished larval chemotaxis towards ethyl acetate. The Orco antagonist was not a general inhibitor of sensory behavior, as behavioral repulsion from a light source was unaffected. This is the first demonstration that an airborne Orco antagonist can alter olfactory behavior in an insect. These results suggest a new approach to insect control and emphasize the need to develop more potent Orco antagonists.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177454PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451006PMC
September 2017

Functional and Nonfunctional Forms of CquiOR91, an Odorant Selectivity Subunit of Culex quinquefasciatus.

Chem Senses 2017 05;42(4):333-341

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, R-189, PO Box 016189, Miami, FL 33101, USA and.

In Culex quinquefasciatus, CquiOR91 is the ortholog of 2 larvae-specific odorant receptors (ORs) from Anopheles gambiae (Agam\Or40, previously shown to respond to several odorant ligands including the broad-spectrum repellent N,N-diethyl-3-methylbenzamide, DEET) and Aedes aegypti (Aaeg\Or40). When we cloned full-length CquiOR91 from a Culex quinquefasciatus larval head RNA sample, we found 2 alleles of this OR, differing at 9 residues. Functional analysis using the Xenopus oocyte expression system and 2-electrode voltage clamp electrophysiology revealed one allele (CquiOR91.1) to be nonfunctional, whereas the other allele (CquiOR91.2) was functional. Receptors formed by CquiOR91.2 and Cqui\Orco responded to (-)-fenchone, (+)-fenchone, and DEET, similar to what has been reported for Agam\Or40. We also identified 5 novel odorant ligands for the CquiOR91.2 + Cqui\Orco receptor: 2-isobutylthiazole, veratrole, eucalyptol, d-camphor, and safranal, with safranal being the most potent. To explore possible reasons for the lack of function for CquiOR91.1, we generated a series of mutant CquiOR91.2 subunits, in which the residue at each of the 9 polymorphic residue positions was changed from what occurs in CquiOR91.2 to what occurs in CquiOR91.1. Eight of the 9 mutant versions of CquiOR91.2 formed functional receptors, responding to (-)-fenchone. Only the CquiOR91.2 Y183C mutant was nonfunctional. The reverse mutation (C183Y) conferred function on CquiOR91.1 , which became responsive to (-)-fenchone and safranal. These results indicate that the "defect" in CquiOR91.1 that prevents function is the cysteine at position 183.
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http://dx.doi.org/10.1093/chemse/bjx011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964367PMC
May 2017

Synthesis, Nicotinic Acetylcholine Receptor Binding, and in Vitro and in Vivo Pharmacological Properties of 2'-Fluoro-(substituted thiophenyl)deschloroepibatidine Analogues.

ACS Chem Neurosci 2017 01 20;8(1):115-127. Epub 2016 Oct 20.

Research Triangle Institute , P.O. Box 12194, Research Triangle Park, North Carolina 27709, United States.

The synthesis, nAChR in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted thiophenyl)deschloroepibatidine analogues (5a-f, 6a-d, and 7a-c) are presented herein. All had subnanomolar affinity at α4β2*-nAChRs. Contrary to lead structure epibatidine, a potent nAChR agonist, all were potent α4β2- and α3β4-AChR antagonists in an in vitro functional assay. In vivo, the compounds were also nAChR antagonists with various degrees of agonist activity. Compounds 5e, 5f, 6a, 6c, 6d, and 7c had no agonist effects in the tail-flick, hot-plate, hypothermia, or spontaneous activity tests, whereas 5a-d, 7a and 7b did not have agonist activity in the tail-flick and hot-plate tests but, like varenicline, were agonists in the hypothermia and spontaneous activity tests. Compound 6b had agonist activity in all four in vivo tests. All the compounds were antagonists of nicotine-induced antinociception in the tail-flick test, and all except 5c, 5d, 5f, and 6b were antagonists of nicotine-induced antinociception in the hot-plate test. Compound 7c, which had a K = 0.86 nM in the binding assay similar potency at α4β2/α3β4 with selectivity relative to α7 nAChRs, had an AD value of 0.001 μg/kg in the tail-flick test with no agonist activity in the in vitro or in vivo test had one of the more interesting profiles.
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http://dx.doi.org/10.1021/acschemneuro.6b00252DOI Listing
January 2017

Synthesis, Nicotinic Acetylcholine Binding, and in Vitro and in Vivo Pharmacological Properties of 2'-Fluoro-(carbamoylpyridinyl)deschloroepibatidine Analogues.

ACS Chem Neurosci 2016 07 19;7(7):1004-12. Epub 2016 May 19.

Research Triangle Institute , P.O. Box 12194, Research Triangle Park, North Carolina 27709, United States.

In this study, we report the synthesis, nAChR in vitro and in vivo pharmacological properties of 2'-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues (5, 6a,b, and 7a,b), which are analogues of our lead structure epibatidine. All of the analogues had subnanomolar binding affinity for α4β2*-nAChRs, and all were potent antagonists of α4β2-nAChRs in an in vitro functional assay. Analogues 6a,b were also highly selective for α4β2- relative to α3β4- and α7-nAChRs. Surprisingly, all of the analogues were exceptionally potent antagonists of nicotine-induced antinociception in the mouse tail-flick test, relative to standard nAChR antagonists such as DHβE. 2'-Fluoro-(4-carbamoyl-3-pyridinyl)deschloroepitabidine (6a) displayed an attractive combination of properties, including subnanomolar binding affinity (Ki = 0.07 nM), submicromolar inhibition of α4β2-nAChRs in the functional assay (IC50 = 0.46 μM) with a high degree of selectivity for α4β2- relative to the α3β4/α7-nAChRs (54-/348-fold, respectively), potent inhibition of [(3)H]dopamine release mediated by α4β2*- and α6β2*-nAChRs in a synaptosomal preparation (IC50 = 21 and 32 nM, respectively), and an AD50 of 0.007 μg/kg as an antagonist of nicotine induced antinociception in the mouse tail-flick test which is 64 250 times more potent than DHβE. These data suggest that compound 6a will be highly useful as a pharmacological tool for studying nAChRs and merits further development.
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http://dx.doi.org/10.1021/acschemneuro.6b00107DOI Listing
July 2016

Synthesis, nicotinic acetylcholine receptor binding, in vitro and in vivo pharmacology properties of 3'-(substituted pyridinyl)-deschloroepibatidine analogs.

Bioorg Med Chem 2015 Sep 17;23(17):5693-701. Epub 2015 Jul 17.

Center for Drug Discovery, Research Triangle Institute, PO Box 12194, Research Triangle Park, NC 27709-2194, United States. Electronic address:

Over the last several years we have synthesized and studied the in vitro and in vivo nAChR pharmacological properties of epibatidine (4) analogs. In this study we report the synthesis, nAChR in vitro and in vivo pharmacological properties of 3'-(substituted pyridinyl)-deschloroepibatidine analogs (5a-e and 6a-e). All of the analogs had high binding affinity for α4β2(∗)-nAChRs. Several of the analogs were potent antagonists of α4β2-nAChRs in in vitro efficacy tests and were potent antagonists of nicotine-induced antinociception in the mouse tail-flick test. Compound 6b had a Ki = 0.13 nM in the binding assay, 25- and 46-fold selectivity for the α4β2(∗)-nAChR relative to the α3β4- and α7-nAChR, respectively, in the in vitro efficacy test and an AD50 = 0.13 μg/kg in the tail-flick test. Combined with favorable calculated physiochemical properties compared to varenicline, our findings suggest that 6b should be considered for development as a potential pharmacotherapy for treating nicotine addiction and other CNS disorders.
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http://dx.doi.org/10.1016/j.bmc.2015.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4554999PMC
September 2015

Proton-dependent glutamine uptake by aphid bacteriocyte amino acid transporter ApGLNT1.

Biochim Biophys Acta 2015 Oct 29;1848(10 Pt A):2085-91. Epub 2015 May 29.

Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. Electronic address:

Aphids house large populations of the gammaproteobacterial symbiont Buchnera aphidicola in specialized bacteriocyte cells. The combined biosynthetic capability of the holobiont (Acyrthosiphon pisum and Buchnera) is sufficient for biosynthesis of all twenty protein coding amino acids, including amino acids that animals alone cannot synthesize; and that are present at low concentrations in A. pisum's plant phloem sap diet. Collaborative holobiont amino acid biosynthesis depends on glutamine import into bacteriocytes, which serves as a nitrogen-rich amino donor for biosynthesis of other amino acids. Recently, we characterized A. pisum glutamine transporter 1 (ApGLNT1), a member of the amino acid/auxin permease family, as the dominant bacteriocyte plasma membrane glutamine transporter. Here we show ApGLNT1 to be structurally and functionally related to mammalian proton-dependent amino acid transporters (PATs 1-4). Using functional expression in Xenopus laevis oocytes, combined with two-electrode voltage clamp electrophysiology we demonstrate that ApGLNT1 is electrogenic and that glutamine induces large inward currents. ApGLNT1 glutamine induced currents are dependent on external glutamine concentration, proton (H+) gradient across the membrane, and membrane potential. Based on these transport properties, ApGLNT1-mediated glutamine uptake into A. pisum bacteriocytes can be regulated by changes in either proton gradients across the plasma membrane or membrane potential.
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http://dx.doi.org/10.1016/j.bbamem.2015.05.019DOI Listing
October 2015

In vitro and in vivo neuronal nicotinic receptor properties of (+)- and (-)-pyrido[3,4]homotropane [(+)- and (-)-PHT]: (+)-PHT is a potent and selective full agonist at α6β2 containing neuronal nicotinic acetylcholine receptors.

ACS Chem Neurosci 2015 Jun 30;6(6):920-6. Epub 2015 Apr 30.

∥Department of Pharmacology, Virginia Commonwealth University Medical Campus, P.O. Box 980615, Richmond, Virginia 23298-0613, United States.

Pyrido[3,4]homotropane (PHT) is a conformationally rigid, high affinity analogue of nicotine. (+)-PHT was previously shown to be 266 times more potent than (-)-PHT for inhibition of [(3)H]epibatidine binding to nAChRs but had no antinociceptive activity in mouse tail-flick or hot-plate tests and was not a nicotinic antagonist even when administered intrathecally. While (-)-PHT had no agonist activity, it was a potent, nicotinic antagonist in the test. Here, electrophysiological studies with rat nAChRs show (+)-PHT to be a low efficacy partial agonist selective for α4β2-nAChRs, relative to α3β4-nAChRs (15-fold) and α7-nAChRs (45-fold). (-)-PHT was an antagonist with selectivity for α3β4, relative to α4β2- (3-fold) and α7- (11-fold) nAChRs. In [(3)H]DA release studies in mice, (+)-PHT was 10-fold more potent than (-)-PHT at α4β2*-nAChRs and 30-fold more potent at α6β2*-nAChRs. Studies using α5KO mice suggested that much of the activity at α4β2*-nAChRs is mediated by the α4β2α5-nAChR subtype. In conditioned place preference studies, (-)-PHT was more potent than (+)-PHT in blocking nicotine reward. Off-target screens showed (+)- and (-)-PHT to be highly selective for nAChRs. The high potency, full agonism of (+)- and (-)-PHT at α6*-nAChR contrasts with the partial agonism observed for α4*-nAChR, making these ligands intriguing probes for learning more about the pharmacophores for various nAChRs.
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http://dx.doi.org/10.1021/acschemneuro.5b00077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589077PMC
June 2015

Receptive range analysis of a mouse odorant receptor subfamily.

J Neurochem 2015 Jul 25;134(1):47-55. Epub 2015 Mar 25.

Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, Florida, USA.

Mammals deploy a large array of odorant receptors (ORs) to detect and distinguish a vast number of odorant molecules. ORs vary widely in the type of odorant structures recognized and in the breadth of molecular receptive range (MRR), with some ORs recognizing a small group of closely related molecules and other ORs recognizing a wide range of structures. While closely related ORs have been shown to have similar MRRs, the functional relationships among less closely related ORs are unclear. We screened a small group of ORs with a diverse odorant panel to identify a new odorant-OR pairing (unsaturated aldehydes and MOR263-3). We then extensively screened MOR263-3 and a series of additional MORs related to MOR263-3 in various ways. MORs related by phylogenetic analysis (several other members of the MOR263 subfamily) had MRRs that overlapped with the MRR of MOR263-3, even with amino acid identity as low as 48% (MOR263-2). MOR171-17, predicted to be functionally related to MOR263-3 by an alternative bioinformatic analysis, but with only 39% amino acid identity, had a distinct odorant specificity. Our results support the use of phylogenetic analysis to predict functional relationships among ORs with relatively low amino acid identity. We screened a small group of mouse odorant receptors (MORs) with a diverse odorant panel to identify a new odorant-OR pairing (unsaturated aldehydes and MOR263-3), then extensively screened a series of additional MORs related to MOR263-3 in various ways. MORs related by phylogenetic analysis had odorant specificities that overlapped with that of MOR263-3, but MOR171-17, predicted to be functionally related to MOR263-3 by an alternative bioinformatic analysis, had a distinct odorant specificity.
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http://dx.doi.org/10.1111/jnc.13095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472571PMC
July 2015

A determinant of odorant specificity is located at the extracellular loop 2-transmembrane domain 4 interface of an Anopheles gambiae odorant receptor subunit.

Chem Senses 2014 Nov 30;39(9):761-9. Epub 2014 Sep 30.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL 33101, USA and

To explore the structural basis for odorant specificity in odorant receptors of the human malaria vector mosquito, Anopheles gambiae, odorant-binding subunits (Agam\Ors) expressed in Xenopus oocytes in combination with Agam\Orco (coreceptor subunit) were assayed by 2-electrode voltage clamp against 25 structurally related odorants. Agam\Or13 and Agam\Or15 display 82% amino acid identity and had similar, but somewhat distinct odorant response profiles. The ratio of acetophenone to 4-methylphenol responses was used in a mutation-based analysis of Agam\Or15, interchanging 37 disparate residues between Agam\Or15 and Agam\Or13. Eleven mutations caused significant changes in odorant responsiveness. Mutation of alanine 195 resulted in the largest shift in response ratio from Agam\Or15 toward Agam\Or13. Concentration-response analysis for a series of mutations of residue 195 revealed a large effect on acetophenone sensitivity, with EC50 values varying by >1800-fold and correlating with residue side chain length. Similar results were obtained for propiophenone and benzaldehyde. But, for other odorants, such as 4-methylphenol, 4-methylbenzaldehyde, and 4-methylpropiophenone, the effect of mutation was much smaller (EC50 values varied by ≤16-fold). These results show that alanine 195, putatively located at the second extracellular loop/fourth transmembrane domain interface, plays a critical role in determining the odorant response specificity of Agam\Or15.
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http://dx.doi.org/10.1093/chemse/bju048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201304PMC
November 2014

Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit.

F1000Res 2014 3;3:84. Epub 2014 Apr 3.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, 33101, USA.

Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control.  Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry.  Synthetic Orco directed agonists and antagonists have recently been identified.  Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants.  The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants.  To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists.  Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system.  Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants.  Tyramine had effects similar to those of tryptamine, but was less potent.  Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae).  Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants.
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http://dx.doi.org/10.12688/f1000research.3825.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097363PMC
July 2014

Discovery of novel ligands for mouse olfactory receptor MOR42-3 using an in silico screening approach and in vitro validation.

PLoS One 2014 17;9(3):e92064. Epub 2014 Mar 17.

Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, Florida, United States of America.

The ligands for many olfactory receptors remain largely unknown despite successful heterologous expression of these receptors. Understanding the molecular receptive range of olfactory receptors and deciphering the olfactory recognition code are hampered by the huge number of odorants and large number of olfactory receptors, as well as the complexity of their combinatorial coding. Here, we present an in silico screening approach to find additional ligands for a mouse olfactory receptor that allows improved definition of its molecular receptive range. A virtual library of 574 odorants was screened against a mouse olfactory receptor MOR42-3. We selected the top 20 candidate ligands using two different scoring functions. These 40 odorant candidate ligands were then tested in vitro using the Xenopus oocyte heterologous expression system and two-electrode voltage clamp electrophysiology. We experimentally confirmed 22 of these ligands. The candidate ligands were screened for both agonist and antagonist activity. In summary, we validated 19 agonists and 3 antagonists. Two of the newly identified antagonists were of low potency. Several previously known ligands (mono- and dicarboxylic acids) are also confirmed in this study. However, some of the newly identified ligands were structurally dissimilar compounds with various functional groups belonging to aldehydes, phenyls, alkenes, esters and ethers. The high positive predictive value of our in silico approach is promising. We believe that this approach can be used for initial deorphanization of olfactory receptors as well as for future comprehensive studies of molecular receptive range of olfactory receptors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092064PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956865PMC
November 2014

Synthesis, nicotinic acetylcholine receptor binding, and antinociceptive properties of 2'-fluoro-3'-(substituted pyridinyl)-7-deschloroepibatidine analogues.

J Med Chem 2014 Feb 28;57(3):836-48. Epub 2014 Jan 28.

Center for Organic and Medicinal Chemistry, Research Triangle Institute , P.O. Box 12194, Research Triangle Park, North Carolina 27709, United States.

2'-Fluoro-3-(substituted pyridine)epibatidine analogues 7a-e and 8a-e were synthesized, and their in vitro and in vivo nAChR properties were determined. 2'-Fluoro-3'-(4″-pyridinyl)deschloroepibatidine (7a) and 2'-fluoro-3'-(3″-pyridinyl)deschloroepibatidine (8a) were synthesized as bioisosteres of the 4'-nitrophenyl lead compounds 5a and 5g. Comparison of the in vitro nAChR properties of 7a and 8a to those of 5a and 5g showed that 7a and 8a had in vitro nAChR properties similar to those of 5a and 5g but both were more selective for the α4β2-nAChR relative to the α3β4- and α7-nAChRs than 5a and 5g. The in vivo nAChR properties in mice of 7a were similar to those of 5a. In contrast, 8a was an agonist in all four mouse acute tests, whereas 5g was active only in a spontaneous activity test. In addition, 5g was a nicotine antagonist in both the tail-flick and hot-plate tests, whereas 8a was an antagonist only in the tail-flick test.
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http://dx.doi.org/10.1021/jm401602pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983394PMC
February 2014

Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts.

Proc Natl Acad Sci U S A 2014 Jan 23;111(1):320-5. Epub 2013 Dec 23.

Department of Biology, University of Miami, Coral Gables, FL 33146.

Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid-Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses.
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http://dx.doi.org/10.1073/pnas.1306068111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890774PMC
January 2014

Phenylthiophenecarboxamide antagonists of the olfactory receptor co-receptor subunit from a mosquito.

PLoS One 2013 17;8(12):e84575. Epub 2013 Dec 17.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

Insects detect environmental chemicals using chemosensory receptors, such as the ORs, a family of odorant-gated ion channels. Insect ORs are multimeric complexes of unknown stoichiometry, formed by a common subunit (the odorant receptor co-receptor subunit, Orco) and one of many variable subunits that confer odorant specificity. The recent discovery of Orco directed ligands, including both agonists and antagonists, suggests Orco as a promising target for chemical control of insects. In addition to competitively inhibiting OR activation by Orco agonists, several Orco antagonists have been shown to act through a non-competitive mechanism to inhibit OR activation by odorants. We previously identified a series of Orco antagonists, including N-(4-ethylphenyl)-2-thiophenecarboxamide (OX1a, previously referred to as OLC20). Here, we explore the chemical space around the OX1a structure to identify more potent Orco antagonists. Cqui\Orco+Cqui\Or21, an OR from Culex quinquefasciatus (the Southern House Mosquito) that responds to 3-methylindole (skatole) and is thought to mediate oviposition behavior, was expressed in Xenopus oocytes and receptor function assayed by two-electrode voltage clamp electrophysiology. 22 structural analogs of OX1a were screened for antagonism of OR activation by an Orco agonist. By varying the moieties decorating the phenyl and thiophene rings, and altering the distance between the rings, we were able to identify antagonists with improved potency. Detailed examination of three of these compounds (N-mesityl-2-thiophenecarboxamide, N-(4-methylbenzyl)-2-thiophenecarboxamide and N-(2-ethylphenyl)-3-(2-thienyl)-2-propenamide) demonstrated competitive inhibition of receptor activation by an Orco agonist and non-competitive inhibition of receptor activation by an odorant. The ability to inhibit OR activation by odorants may be a general property of this class of Orco antagonist, suggesting that odorant mediated behaviors can be manipulated through Orco antagonism. The high conservation of Orco across insect species and previous demonstrations that various Orco ligands are active at ORs derived from several different insect orders suggests that Orco antagonists may have broad applicability.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084575PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866151PMC
September 2014

Silent, generic and plant kairomone sensitive odorant receptors from the Southern house mosquito.

J Insect Physiol 2013 Sep 19;59(9):961-6. Epub 2013 Jul 19.

Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.

The Southern house mosquito Culex quinquefasciatus has the largest repertoire of odorant receptors (ORs) of all mosquitoes and dipteran species whose genomes have been sequenced to date. Previously, we have identified and de-orphanized two ORs expressed in female antennae, CquiOR2 and CquiOR10, which are sensitive to oviposition attractants. In view of a new nomenclature for the Culex genome (VectorBase) we renamed these ORs as CquiOR21 (formerly CquiOR10) and CquiOR121 (CquiOR2). In addition, we selected ORs from six different phylogenetic groups for deorphanization. We cloned four of them by using cDNA from female antennae as a template. Attempts to clone CquiOR87 and CquiOR110 were unsuccessful either because they are pseudogenes or are not expressed in adult female antennae, the main olfactory tissue. By contrast, CquiOR1, CquiOR44, CquiOR73, and CquiOR161 were highly expressed in female antennae. To de-orphanize these ORs, we employed the Xenopus oocyte recording system. CquiORx-CquiOrco-expressed oocytes were challenged with a panel of 90 compounds, including known oviposition attractants, human and vertebrate host odorants, plant kairomones, and naturally occurring repellents. While CquiOR161 did not respond to any test compound in two different laboratories, CquiOR1 showed the features of a generic OR, with strong responses to 1-octen-3-ol and other ligands. CquiOR44 and CquiOR73 showed preference to plant-derived terpenoids and phenolic compounds, respectively. While fenchone was the best ligand for the former, 3,5-dimethylphenol elicited the strongest responses in the latter. The newly de-orphanized ORs may be involved in reception of plant kairomones and/or natural repellents.
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http://dx.doi.org/10.1016/j.jinsphys.2013.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3800014PMC
September 2013

Functional assay of mammalian and insect olfactory receptors using Xenopus oocytes.

Methods Mol Biol 2013 ;1003:187-202

Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL, USA.

The large number of olfactory receptors (ORs) expressed by various mammalian and insect species, as well as the large number of potential odorant ligands, has made the pairing of odorants with receptors -(de-orphaning) exceedingly difficult. These efforts are further complicated by difficulties in expressing ORs in many standard expression systems. Xenopus laevis oocytes offer a versatile expression platform for the de-orphaning and functional characterization of ORs. Two-electrode voltage clamp electrophysiology is a common and relatively straightforward approach to the functional assay of receptors expressed in Xenopus oocytes, and this technique has been discussed extensively in the literature. However, laboratories that are new to the use of Xenopus oocytes are often stymied by some of the peculiarities of the Xenopus oocyte expression system. We discuss some of the key methodological issues in Xenopus care, oocyte -isolation and receptor expression, with a focus on using this expression system to study the ORs of mammals and insects.
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http://dx.doi.org/10.1007/978-1-62703-377-0_14DOI Listing
September 2013

Receptors: Chemical courtship in mice.

Authors:
Charles W Luetje

Nat Chem Biol 2013 Mar;9(3):140-1

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http://dx.doi.org/10.1038/nchembio.1180DOI Listing
March 2013

Synthesis and nicotinic acetylcholine receptor in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted phenyl)deschloroepibatidine analogues of 2'-fluoro-3'-(4-nitrophenyl)deschloroepibatidine.

J Med Chem 2012 Jul 11;55(14):6512-22. Epub 2012 Jul 11.

Center for Organic and Medicinal Chemistry, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC 27709, USA.

Herein, we report the synthesis and nicotinic acetylcholine receptor (nAChR) in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines 5b-g, analogues of 3'-(4-nitrophenyl) compound 5a. All compounds had high affinity for α4β2-nAChR and low affinity for α7-nAChR. Initial electrophysiological studies showed that all analogues were antagonists at α4β2-, α3β4-, and α7-nAChRs. The 4-carbamoylphenyl analogue 5g was highly selective for α4β2-nAChR over α3β4- and α7-nAChRs. All the analogues were antagonists of nicotine-induced antinociception in the tail-flick test. Molecular modeling docking studies using the agonist-bound form of the X-ray crystal structure of the acetylcholine binding protein suggested several different binding modes for epibatidine, varenicline, and 5a-g. In particular, a unique binding mode for 5g was suggested by these docking simulations. The high binding affinity, in vitro efficacy, and selectivity of 5g for α4β2-nAChR combined with its nAChR functional antagonist properties suggest that 5g will be a valuable pharmacological tool for studying the nAChR and may have potential as a pharmacotherapy for addiction and other central nervous system disorders.
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http://dx.doi.org/10.1021/jm300575yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431023PMC
July 2012

Identification of new agonists and antagonists of the insect odorant receptor co-receptor subunit.

PLoS One 2012 8;7(5):e36784. Epub 2012 May 8.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

Background: Insects detect attractive and aversive chemicals using several families of chemosensory receptors, including the OR family of olfactory receptors, making these receptors appealing targets for the control of insects. Insect ORs are odorant-gated ion channels, comprised of at least one common subunit (the odorant receptor co-receptor subunit, Orco) and at least one variable odorant specificity subunit. Each of the many ORs of an insect species is activated or inhibited by an unique set of odorants that interact with the variable odorant specificity subunits, making the development of OR directed insect control agents complex and laborious. However, several N-,2-substituted triazolothioacetamide compounds (VUAA1, VU0450667 and VU0183254) were recently shown to act directly on the highly conserved Orco subunit, suggesting that broadly active compounds can be developed. We have explored the chemical space around the VUAA1 structure in order to identify new Orco ligands.

Principal Findings: We screened ORs from several insect species, using heterologous expression in Xenopus oocytes and an electrophysiological assay, with a panel of 22 compounds structurally related to VUAA1. By varying the nitrogen position in the pyridine ring and altering the moieties decorating the phenyl ring, we identified two new agonists and a series of competitive antagonists. Screening smaller compounds, similar to portions of the VUAA1 structure, also yielded competitive antagonists. Importantly, we show that Orco antagonists inhibit odorant activation of ORs from several insect species. Detailed examination of one antagonist demonstrated inhibition to be through a non-competitive mechanism.

Conclusions: A similar pattern of agonist and antagonist sensitivity displayed by Orco subunits from different species suggests a highly conserved binding site structure. The susceptibility to inhibition of odorant activation by Orco antagonism is conserved across disparate insect species, suggesing the ligand binding site on Orco as a promising target for the development of novel, broadly active insect repellants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036784PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348135PMC
September 2012

Sequencing and characterizing odorant receptors of the cerambycid beetle Megacyllene caryae.

Insect Biochem Mol Biol 2012 Jul 5;42(7):499-505. Epub 2012 Apr 5.

Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Odorant receptors (Ors) are a unique family of ligand-gated ion channels and the primary mechanism by which insects detect volatile chemicals. Here, we describe 57 putative Ors sequenced from an antennal transcriptome of the cerambycid beetle Megacyllene caryae (Gahan). The male beetles produce a pheromone blend of nine compovnents, and we functionally characterized Ors tuned to three of these chemicals: receptor McOr3 is sensitive to (S)-2-methyl-1-butanol; McOr20 is sensitive to (2S,3R)-2,3-hexanediol; and McOr5 is sensitive to 2-phenylethanol. McOr3 and McOr20 are also sensitive to structurally-related chemicals that are pheromones of other cerambycid beetles, suggesting that orthologous receptors may be present across many cerambycid species. These Ors are the first to be functionally characterized from any species of beetle and lay the groundwork for understanding the evolution of pheromones within the Cerambycidae.
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http://dx.doi.org/10.1016/j.ibmb.2012.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361640PMC
July 2012

A broadly tuned mouse odorant receptor that detects nitrotoluenes.

J Neurochem 2012 Jun 19;121(6):881-90. Epub 2012 Apr 19.

Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, Florida 33101, USA.

Mammals employ large numbers of odorant receptors to sample and identify volatile chemicals in the environment. These receptors are thought to vary not only in specificity for particular odorants, but also in breadth of tuning. That is, some odorant receptors are narrowly focused on a few closely related structures, while other odorant receptors may be 'broadly tuned', responding to a wide variety of odorant structures. In this study, we have performed a detailed examination the mouse odorant receptor MOR256-17, demonstrating that this receptor is broadly tuned. This receptor responds to odorant structures that span a significant portion of a multi-dimensional odor space. However, we found that broad tuning was not a defining characteristic of other members the MOR256 subfamily. Two additional members of this odorant receptor subfamily (MOR256-8 and MOR256-22) were more narrowly focused on small sets of odorant structures. Interestingly, the receptive range of MOR256-17 encompassed a variety of nitrotoluenes, including various trinitrotoluene synthesis intermediates, degradation products and trinitrotoluene itself, suggesting the potential utility of odorant receptors in the development of sensing technologies for the detection of explosives and other forms of contraband.
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http://dx.doi.org/10.1111/j.1471-4159.2012.07740.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3371092PMC
June 2012

RegIIA: an α4/7-conotoxin from the venom of Conus regius that potently blocks α3β4 nAChRs.

Biochem Pharmacol 2012 Feb 16;83(3):419-26. Epub 2011 Nov 16.

Department of Chemistry & Biochemistry, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL 33431, USA.

Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinson's disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. α-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/α-conotoxin pair under consideration. Thus, α-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes. We isolated an α4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage clamp recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the α3β4 nAChRs found to date and is also active at α3β2 and α7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most α4/7-conotoxins. Thus, while structurally related to other α4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the α3β4 nAChRs.
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http://dx.doi.org/10.1016/j.bcp.2011.11.006DOI Listing
February 2012

Biomimetic chemical sensors using nanoelectronic readout of olfactory receptor proteins.

ACS Nano 2011 Jul 6;5(7):5408-16. Epub 2011 Jul 6.

Department of Physics & Astronomy, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

We have designed and implemented a practical nanoelectronic interface to G-protein coupled receptors (GPCRs), a large family of membrane proteins whose roles in the detection of molecules outside eukaryotic cells make them important pharmaceutical targets. Specifically, we have coupled olfactory receptor proteins (ORs) with carbon nanotube transistors. The resulting devices transduce signals associated with odorant binding to ORs in the gas phase under ambient conditions and show responses that are in excellent agreement with results from established assays for OR-ligand binding. The work represents significant progress on a path toward a bioelectronic nose that can be directly compared to biological olfactory systems as well as a general method for the study of GPCR function in multiple domains using electronic readout.
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http://dx.doi.org/10.1021/nn200489jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4165341PMC
July 2011

Subunit contributions to insect olfactory receptor function: channel block and odorant recognition.

Chem Senses 2011 Nov 15;36(9):781-90. Epub 2011 Jun 15.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, FL 33101, USA.

Insect olfactory receptors are heteromeric ligand-gated ion channels composed of at least one common subunit (Orco) and at least one subunit that confers odorant specificity. Little is known about how individual subunits contribute to the structure and function of the olfactory receptor complex. We expressed insect olfactory receptors in Xenopus oocytes to investigate 2 functional features, ion channel block and odorant recognition. The sensitivity of Drosophila olfactory receptors to inhibition by ruthenium red, a cation channel blocker, varied widely when different specificity subunits were present, suggesting that the specificity subunits contribute to the structure of the ion pore. Olfactory receptors formed by Dmel\Or35a and Orco subunits from several different species displayed highly similar odorant response profiles, suggesting that the Orco subunit does not contribute to the structure of the odorant-binding site. We further explored odorant recognition by conducting a detailed examination of the odorant specificity Dmel\Or67a + Dmel\Orco, a receptor that responds to aromatic structures. This screen identified agonists, partial agonists, and an antagonist of Dmel\Or67a + Dmel\Orco. Our findings favor specific subunit arrangements within the olfactory receptor complex and provide a preliminary odorophore for an olfactory receptor, offering a useful foundation for future exploration of insect olfactory receptor structure.
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http://dx.doi.org/10.1093/chemse/bjr053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195787PMC
November 2011

Odorant receptor from the southern house mosquito narrowly tuned to the oviposition attractant skatole.

J Chem Ecol 2010 Aug 11;36(8):797-800. Epub 2010 Jul 11.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, USA.

Oviposition attractants are environmental cues that allow Culex gravid female mosquitoes to locate suitable sites for egg-laying and, therefore, may be exploited for environmentally friendly strategies for controlling mosquito populations. Naturally occurring skatole has been identified as an oviposition attractant for the Southern House mosquito, Culex quinquefasciatus. Previously, we identified in Cx. quinquefasciatus female antennae an olfactory receptor neuron (ORN) highly sensitive to skatole and an odorant-binding protein involved in the detection of this semiochemical. Here, we describe the characterization of an odorant receptor (OR), CquiOR10, which is narrowly tuned to skatole when expressed in the Xenopus oocyte system. Odorant-induced response profiles generated by heterologously expressed CquiOR10 suggest that this OR is expressed in the mosquito ORN sensitive to skatole. However, geranylacetone, which stimulates the antennal ORN, was not detected by CquiOR10-expressing oocytes, thus raising interesting questions about reception of oviposition attractants in mosquitoes.
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http://dx.doi.org/10.1007/s10886-010-9828-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908433PMC
August 2010