An Odorant Receptor from the Southern House Mosquito Culex pipiens quinquefasciatus Sensitive to Oviposition Attractants

Insect odorant receptors (ORs) are heteromers comprised of highly variable odorant-binding subunits associated with one conserved co-receptor. They are potential molecular targets for the development of novel mosquito attractants and repellents. ORs have been identified in the malaria mosquito, Anopheles gambiae, and in the yellow fever mosquito, Aedes aegypti. However, they are still unknown in the Southern house mosquito, Culex quinquefasciatus, which transmits pathogens that cause human diseases throughout the world, including West Nile Virus in the United States.We have employed a combination of bioinformatics, molecular cloning and electrophysiology approaches to identify and characterize the response profile of an OR in Cx. quinquefasciatus. First, we have unveiled a large multigenic family of one-hundred-fifty-eight putative ORs in this species, including a subgroup of conserved ORs in three mosquito species. Using the Xenopus oocytes expression system, we have determined the response profile of CquiOR2, an antennae-specific OR, which shares high identity with putative orthologs in Anopheles gambiae (AgamOR2) and Aedes aegypti (AaegOR2).We show that CquiOR2 is highly sensitive to indole, an oviposition attractant for Cx. quinquefasciatus. The response profile of CquiOR2 expressed in Xenopus oocytes resembles that of an olfactory receptor neuron housed in the antennal short blunt-tipped sensilla (A2) of Cx. quinquefasciatus, which are natural detectors for oviposition attractants. This first Culex OR de-orphanized is, therefore, a potential molecular target for screening oviposition attractants

Heterogeneity of Nicotinic Receptor Class and Subunit mRNA Expression among Individual Parasympathetic Neurons from Rat Intracardiac Ganglia

Neurons have the potential to form thousands of distinct neuronal nicotinic receptors from the eight alpha and three beta subunits that currently are known. In an effort to determine how much of this potential complexity is realized among individual neurons, we examined the nicotinic pharmacological and biophysical properties and receptor subunit mRNA expression patterns in individual neurons cultured from rat epicardial ganglia. Analysis of the whole-cell pharmacology of these neurons showed a diversity of responses to the agonists acetylcholine, nicotine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium, suggesting that a heterogeneous population of nicotinic receptor classes, or subtypes, is expressed by individual neurons. Single-channel analysis demonstrated three distinct conductances (18, 24, and 31 pS), with patches from different neurons containing different combinations of these channel classes. We used single-cell RT-PCR to examine nicotinic acetylcholine receptor (nAChR) subunit mRNA expression by individual neurons. Although mRNAs encoding all eight neuronal nAChR subunits for which we probed (alpha 2-alpha 5, alpha 7, beta 2-beta 4) were present in multicellular cultures, we found that individual epicardial neurons express distinct subsets of these nAChR subunit mRNAs. These results suggest that individual epicardial neurons express distinct arrays of nAChR subunits and that these subunits may assemble into functional receptors with distinct and variable subunit composition. This variable receptor subunit expression provides an explanation for the diversity of pharmacological and single-channel responses we have observed in individual neurons

Differential Roles for Disulfide Bonds in the Structural Integrity and Biological Activity of κ-Bungarotoxin, a Neuronal Nicotinic Acetylcholine Receptor Antagonist †

ABSTRACT: κ-Bungarotoxin, a κ-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. κ-Bungarotoxin is a potent, selective, and slowly reversible antagonist of R3 2 neuronal nicotinic acetylcholine receptors. κ-Bungarotoxin is structurally related to the R-neurotoxins, such as R-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (R1 1γδ) and the homopentameric neuronal type receptors (R7, R8, and R9). Like the κ-neurotoxins, the long R-neurotoxins contain the same five conserved disulfide bonds, while the short R-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in κ-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors. The R-and κ-neurotoxins, isolated from the venoms of snakes from the elapid and hydrophid families, are effective antagonists of nicotinic acetylcholine receptors (1). While the R-neurotoxins effectively antagonize muscle type receptors (R1 1γδ) and the homopentameric neuronal type receptors (R7, R8, and R9), the κ-neurotoxins are potent, selective antagonists of R3 2 (and to a lesser extent R4 2) neuronal nicotinic receptors (2). The binding to these receptors is characterized by very slow kinetic off rates for both classes of toxin. It has also been reported that κ-bungarotoxin is capable of inhibiting receptors containing R2 2, R2 4, R3 4, R4 4, and even R1 1γδ combinations expressed in frog oocytes when the toxin is coapplied with agonist (3). In this case, however, the kinetics of inhibition is characterized by very fast off rates. The reason for these differences in kinetic behavior is currently unknown. In addition to their characteristic interactions with receptors, the R-and κ-neurotoxins also display distinctive structural characteristics. Prominent among these is the observation that the R-neurotoxins are monomeric in solution while the κ-neurotoxins are dimeric in solution. The dimerization of κ-bungarotoxin has been shown to result from specific residues which are conserved in the κ-neurotoxins but not the R-neurotoxins that interact at a dimer interface formed by the two strands of the third loop of each subunit (4). The dimeric nature of the κ-neurotoxins may play a role in their binding to receptors and may account at least in part for their different specificity and kinetic characteristics (5). These neurotoxins can also be classified as either short or long neurotoxins based on conserved elements of structure. Short neurotoxins contain 60-62 amino acid residues and four disulfide bridges in common positions. Long neurotoxins usually consist of 66-74 amino acid residues and have a fifth disulfide bond in addition to the four found in short neurotoxins. Short and long neurotoxins are also distinguished by conserved sequence deletions and additions relative to one another (1). Both short and long neurotoxins, including κ-neurotoxins, have been studied extensively by site-directed mutagenesis (6-9), but there have been few reports regarding the function or role of the conserved disulfide bonds. Do these bonds simply play a structural role in stabilizing the conformation

Odorant Receptor from the Southern House Mosquito Narrowly Tuned to the Oviposition Attractant Skatole

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

Genetic Elucidation of Human Hyperosmia to Isovaleric Acid

The genetic basis of odorant-specific variations in human olfactory thresholds, and in particular of enhanced odorant sensitivity (hyperosmia), remains largely unknown. Olfactory receptor (OR) segregating pseudogenes, displaying both functional and nonfunctional alleles in humans, are excellent candidates to underlie these differences in olfactory sensitivity. To explore this hypothesis, we examined the association between olfactory detection threshold phenotypes of four odorants and segregating pseudogene genotypes of 43 ORs genome-wide. A strong association signal was observed between the single nucleotide polymorphism variants in OR11H7P and sensitivity to the odorant isovaleric acid. This association was largely due to the low frequency of homozygous pseudogenized genotype in individuals with specific hyperosmia to this odorant, implying a possible functional role of OR11H7P in isovaleric acid detection. This predicted receptor–ligand functional relationship was further verified using the Xenopus oocyte expression system, whereby the intact allele of OR11H7P exhibited a response to isovaleric acid. Notably, we also uncovered another mechanism affecting general olfactory acuity that manifested as a significant inter-odorant threshold concordance, resulting in an overrepresentation of individuals who were hyperosmic to several odorants. An involvement of polymorphisms in other downstream transduction genes is one possible explanation for this observation. Thus, human hyperosmia to isovaleric acid is a complex trait, contributed to by both receptor and other mechanisms in the olfactory signaling pathway

Identification of New Agonists and Antagonists of the Insect Odorant Receptor Co-Receptor Subunit

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

2002. Modulation of neuronal nicotinic acetylcholine receptors by mercury

ABSTRACT Mercuric chloride exerted a biphasic modulatory effect on rat neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes as heteromers of the ␣3 or ␣4 and ␤2 or ␤4 subunits. The degree of modulation was subunit-dependent, with ␤4-containing receptors displaying greater potentiation and ␣4-containing receptors displaying greater inhibition. Thus, ␣4␤4 receptors displayed both robust potentiation and robust inhibition. During prolonged coapplication of HgCl 2 , first potentiation then inhibition of the acetylcholine (ACh) response was observed. Upon coapplication of 1 M HgCl 2 , a 2-fold increase in ACh-induced current was achieved in 55 Ϯ 1 s. With continued HgCl 2 application, the ACh response was slowly inhibited until, after 5 min, less than 10% of the initial response remained. By measuring potentiation at its peak and inhibition 5 min after the start of HgCl 2 coapplication, we obtained EC 50 and IC 50 values of 262 Ϯ 75 and 430 Ϯ 72 nM, respectively. HgCl 2 potentiation was voltage-dependent, increasing at more positive holding potentials. Upon washout of mercury chloride, potentiation reversed with a t 1/2 of 4.6 min. Inhibition reversed more slowly, with less than half the initial response recovered after 15 min of wash. Although free cysteine residues are common targets for mercury, elimination of all free cysteines located in the extracellular domains of the ␣4 and ␤4 subunits did not alter the effects of mercuric chloride. Potentiation and inhibition of neuronal nAChRs may occur through action at a transmembrane or cytoplasmic location after passive diffusion of mercuric chloride across the plasma membrane

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

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