Chloride Channel Subunit Research Articles

Teladorsagia circumcincta: Molecular characterisation of the avr-14B subunit and its relatively minor role in ivermectin resistance

Individual mutations (e.g. L256F) and polymorphisms in the avr-14B gene, a glutamate-gated chloride channel subunit, have been associated with ivermectin (IVM) resistance in Caenorhabditis elegans and Cooperia oncophora. The aim of the present study was to determine the full-length coding sequence of the avr-14B subunit homologue in Teladorsagia circumcincta and determine the presence/absence of the putative L256F SNP or any other potential SNPs of interest. Subsequently, we investigated sequence polymorphisms and transcription patterns between four different T. circumcincta isolates: two from Scotland (MTci1 susceptible and MTci5 triple resistant to benzimidazoles, levamisole and IVM) and two from Spain (S-Sp susceptible and R-Sp double resistant to levamisole and IVM). The complete amino acid sequence of the T. circumcincta avr-14B subunit comprises 438 amino acids. Pyrosequencing analysis failed to detect the presence of the L256F mutation in any of the MTci5 or Sp-R samples tested. However, we revealed significant allele frequency changes by means of SSCP analysis of a 106bp region encompassing the L256F SNP. Allele E showed the greatest change, following IVM exposure in vitro and in vivo, although sequence analysis did not reveal any coding changes. Sequence analysis of the full-length avr-14B coding sequence showed that two SNPs exclusively found in the resistant strain McTi5 (I270F and T305A) are situated in codons involved in the interaction of the receptor with IVM. Moreover, other potentially significant SNPs (K361E and L364M) were identified between transmembrane regions 3 and 4. However, due to the low frequency of all these SNPs, we cannot conclude they confer IVM resistance in T. circumcincta. Moreover, a modest increase in expression of the avr-14B in both resistant isolates has been shown although these differences were not sufficiently great to consider avr-14B to be the sole or even a major determinant of IVM resistance in this species.

Mechanisms Underlying Fipronil Resistance in a Multiresistant Field Strain of the German Cockroach (Blattodea: Blattellidae)

German cockroaches (Blattella germanica L.) have significant impacts on human health, most notably they are implicated as causes of childhood asthma. Gel bait formulations of fipronil, a phenylpyrazole insecticide, have been in use for German cockroach control in the United States since 1998. Previously, dieldrin resistant German cockroach strains were shown to have 7- to 17-fold cross-resistance to fipronil. More recently, a field-collected strain (GNV-R) displayed approximately 36-fold resistance to topically applied fipronil at the LD50 level, which is the highest level of fipronil resistance reported to date in the German cockroach. The aim of the current research was to identify mechanism(s) responsible for high-level fipronil resistance in the GNV-R strain. Synergist bioassays conducted using topical and injection application methods implicated cytochrome P450-mediated detoxification in resistance. Electrophysiological recordings using the suction-electrode technique revealed the nervous system of the GNV-R strain is insensitive to fipronil. In agreement with electrophysiology results, the alanine to serine (A302S) mutation encoded by the gamma-amino butyric acid-gated chloride channel subunit gene resistance to dieldrin, which confers limited cross-resistance to fipronil, was detected in 95% of GNV-R strain individuals. Logistic regression analysis showed that A302S mutation frequency correlates with neurological insensitivity as shown by electrophysiology data. Overall, results of synergism bioassays, electrophysiological recordings, and A302S mutation frequency measurements suggest that fipronil resistance in the GNV-R strain is caused by the combined effects of enhanced metabolism by cytochrome P450s and target-site insensitivity caused by the A302S-encoding mutation in the resistance to dieldrin gene.

Candidate anthelmintic resistance-associated gene expression and sequence polymorphisms in a triple-resistant field isolate of Haemonchus contortus

An isolate of Haemonchus contortus, UGA/2004, highly resistant to benzimidazoles, levamisole, and ivermectin was isolated from sheep at the University of Georgia, and passaged through experimentally infected goats. We measured the expression of twenty-nine mRNAs encoding drug targets and P-glycoproteins (P-gps), comparing the results to a fully susceptible laboratory passaged isolate. Expression levels of some nicotinic acetylcholine receptor mRNAs were markedly different in UGA/2004. Levels of the Hco-acr-8b mRNA, encoding a truncated subunit, were very high in resistant L3, but undetectable in susceptible larvae, with expression of the full-length Hco-acr-8a mRNA also significant increased. Expression of Hco-unc-63 and Hco-unc-29.3 mRNAs was significantly reduced in the resistant larvae. Expression of the Hco-glc-3 and Hco-glc-5 mRNAs, encoding glutamate-gated chloride channel subunits, were slightly reduced in resistant larvae. We observed significant increases in the expression of the Hco-pgp-2 and Hco-pgp-9 mRNAs in the UGA/2004 larvae, consistent with previous reports; we also saw a decrease in the levels of Hco-pgp-1 mRNA. Treatment of the larvae with ivermectin and moxidectin in vitro produced variable and inconsistent changes in P-gp mRNA levels. The sequences of the β-tubulin isotype 1 mRNAs showed that the resistant larvae had a resistance-associated allele frequency of >95% at codon 200 and ∼40% and codon 167. No changes at codon 198 were present. The presence of the truncated acr-8b mRNA may be a reliable indicator of levamisole resistance, but complex changes in gene expression associated with macrocyclic lactone resistance make the identification of a single genetic marker for this resistance difficult.

An UNC‐49 GABA receptor subunit from the parasitic nematode Haemonchus contortus is associated with enhanced GABA sensitivity in nematode heteromeric channels

We have identified two genes from the parasitic nematode Haemonchus contortus, Hco-unc-49B and Hco-unc-49C that encode two GABA-gated chloride channel subunits. Electrophysiological analysis revealed that this channel has properties similar to those of the UNC-49 channel from the free-living nematode Caenorhabditis elegans. For example, the Hco-UNC-49B subunit forms a functional homomeric channel that responds to GABA and is highly sensitive to picrotoxin. Hco-UNC-49C alone does not respond to GABA but can assemble with Hco-UNC-49B to form a heteromeric channel with a lower sensitivity to picrotoxin. However, we did find that the Hco-UNC-49B/C heteromeric channel is significantly more responsive to agonists compared to the Hco-UNC-49B homomeric channel, which is the opposite trend to what has been found previously for the C. elegans channel. To investigate the subunit requirements for high agonist sensitivity, we generated cross-assembled channels by co-expressing the H. contortus subunits with UNC-49 subunits from C. elegans (Cel-UNC-49). Co-expressing Cel-UNC-49B with Hco-UNC-49C produced a heteromeric channel with a reduced sensitivity to GABA compared to that of the Cel-UNC-49B homomeric channel. In contrast, co-expressing Hco-UNC-49B with Cel-UNC-49C produced a heteromeric channel that, like the Hco-UNC-49B/C heteromeric channel, exhibits an increased sensitivity to GABA. These results suggest that the Hco-UNC-49B subunit is the key determinant for the high agonist sensitivity of heteromeric channels.

Evidence supporting the existence of a novel histaminergic pathway in the regulation of EAE susceptibility (143.51)

Abstract Histamine (HA) is a key regulator of experimental allergic encephalomyelitis (EAE), the autoimmune disease model of multiple sclerosis (MS). Histidine decarboxylase deficient mice (HDCKO), which are unable to synthesize HA, exhibit more severe EAE and increased IFN-γ production by splenocytes in response to MOG35-55. HA exerts its effects through four different G protein coupled receptors (GPCR): H1, H2, H3 and H4 (H1-4R). Each HA-receptor has been shown to influence EAE pathogenesis. In the mammalian brain there is, however, evidence for the existence of non-GPCR signaling by HA which is picrotoxin-sensitive and mediated by chloride conductance. GABAA receptor subunits can form HA-gated chloride channels in vitro suggesting that an ionotropic HA-receptor might contain known ligand-gated chloride channel subunits. To test the hypothesis that non-GPCR signaling by HA plays a role in immune responses, we generated H1-4RKO mice and studied their susceptibility to EAE. Here we report that in contrast to HDCKO mice, H1-4RKO mice develop less severe EAE compared to WT animals. Furthermore, splenocytes from immunized H1-4RKO mice produce significantly less IFN-γ compared to WT mice. Taken together these data support the existence of a novel HA signaling pathway in regulating EAE susceptibility.

A Case of Antenatal Bartter Syndrome with Sensorineural Deafness

Bartter syndrome type IV, also known as Bartter syndrome with sensorineural deafness (BSND), is caused by loss-of-function mutations in the BSND gene, which encodes barttin, an accessory subunit of chloride channels located in the kidney and inner ear. Patients with BS IV have a highly variable clinical phenotype. This report concerns a Korean male patient with antenatal Bartter syndrome due to a homozygous BSND p.G47R mutation, who presented with severe perinatal symptoms followed by a relatively benign course with preserved renal function after early infancy. In addition, the clinical features and the laboratory data of the patient were compared with those of previously reported patients with the same mutation.

The actions of chloride channel blockers, barbiturates and a benzodiazepine on Caenorhabditis elegans glutamate- and ivermectin-gated chloride channel subunits expressed in Xenopus oocytes

The pharmacology of Caenorhabditis elegans glutamate-gated chloride (GluCl) channels was determined by making intracellular voltage-clamp recordings from Xenopus oocytes expressing GluCl subunits. As previously reported (Cully et al. 1994), GluClalpha1beta responded to glutamate (in a picrotoxin sensitive manner) and ivermectin, while GluClbeta responded only to glutamate and GluClalpha1 only to ivermectin. This assay was used to further investigate the action of chloride channel compounds. The arylaminobenzoate, NPPB, reduced the action of glutamate on the heteromeric GluClalpha1beta channel (IC(50) 6.03 +/- 0.81 microM). The disulphonate stilbene, DNDS, blocked the effect of both glutamate and ivermectin on GluClalpha1beta channels, the action of glutamate on GluClbeta subunits, and the effect of ivermectin on GluClalpha1 subunits (IC(50)s 1.58-3.83 microM). Surprisingly, amobarbital and pentobarbital, otherwise known as positive allosteric modulators of ligand-gated chloride channels, acted as antagonists. Both compounds reduced the action of glutamate on the GluClalpha1beta heteromer (IC(50)s of 2.04 +/- 0.5 and 17.56 +/- 2.16 microM, respectively). Pentobarbital reduced the action of glutamate on the GluClbeta homomeric subunit with an IC(50) of 0.59 +/- 0.09 microM, while reducing the responses to ivermectin on both GluClalpha1beta and GluClalpha1 with IC(50)s of 8.7 +/- 0.5 and 12.9 +/- 2.5 microM, respectively. For all the antagonists, the mechanism is apparently non-competitive. The benzodiazepine, flurazepam had no apparent effect on these glutamate- and ivermectin-gated chloride channel subunits. Thus, arylaminobenzoates, disulphonate stilbenes, and barbiturates are non-competitive antagonists of C. elegans GluCl channels.

The ligand-gated chloride channel gene family of Drosophila melanogaster

The antagonism of cellular excitability in insects is mediated by a family of ionotropic receptors, the ligand-gated chloride channels. In insects these inhibitory receptors include synaptic GABA and histamine receptors, functionally analogous to vertebrate GABA and glycine receptors, and glutamate receptors that appear to be unique to invertebrates. The ligand-gated chloride channel family in insects includes two well-validated targets for insect control agents: GABA receptors, which are the targets for a structural variety of small-molecule insecticides (polychlorocycloalkanes such as dieldrin and phenylpyrazoles such as fipronil) that have been used widely in agriculture; and glutamate receptors, which are the targets of macrocyclic lactone natural products (the avermectins) that have yielded not only commercial insecticides but also anthelminthic agents (i.e., ivermectin) employed in animal and human health. The availability of the complete genome sequence of the insect model system Drosophila melanogaster has permitted the identification of all of the genes encoding proteins with structural similarity to known ligand-gated chloride channel subunits (ligand-gated chloride channel homologs or LCCHs). Here, we review the present status of knowledge of the structure and function of the proteins encoded by this compact gene family. The 12 LCCH genes of D. melanogaster exhibit a surprising degree of structural diversity, which is further enhanced for some subunits by a variety of post-transcriptional and post-translational modifications. Although the structures of the gene products encoded by this small gene family are now well characterized, surprisingly little is known of the biological functions of the majority of them and the structures of most native receptors remain unknown.

Functional consequences of mutations in the Drosophila histamine receptor HCLB

The gene hclB encodes a histamine-gated chloride channel subunit in Drosophila melanogaster. Mutations in hclB lead to defects in the visual system and altered sensitivity to the action of ivermectin. To investigate whether this member of the Cys-loop receptors is common across the Insecta, we analysed the genomes of seven other insect species (Diptera, Hymenoptera, Coleoptera) and revealed orthologues of hclB in all of them. Sequence comparisons showed high identity levels between the orthologues, indicating similar constraints and conserved function between the species. Two D. melanogaster mutants, hclBT1 (P293S) and hclBT2 (W111*, a null mutation) were tested for the lapse into, and recovery from, paralysis induced by high temperature or the anaesthetic action of halothane. At 41°C, the hclBT2 flies lapsed into coma faster than wild-type or the hclBT1 flies, while both mutants recovered more slowly. A substantially impaired recovery rate was also observed in hclBT1 after anaesthesia with halothane. Enhanced synaptic signalling at low-intensity light stimuli was registered on electroretinograms recorded from the two mutant strains. Our results suggest that HCLB may play an essential and conserved role in insect neurophysiology.

Molecular Basis of DFNB73: Mutations of BSND Can Cause Nonsyndromic Deafness or Bartter Syndrome

BSND encodes barttin, an accessory subunit of renal and inner ear chloride channels. To date, all mutations of BSND have been shown to cause Bartter syndrome type IV, characterized by significant renal abnormalities and deafness. We identified a BSND mutation (p.I12T) in four kindreds segregating nonsyndromic deafness linked to a 4.04-cM interval on chromosome 1p32.3. The functional consequences of p.I12T differ from BSND mutations that cause renal failure and deafness in Bartter syndrome type IV. p.I12T leaves chloride channel function unaffected and only interferes with chaperone function of barttin in intracellular trafficking. This study provides functional data implicating a hypomorphic allele of BSND as a cause of apparent nonsyndromic deafness. We demonstrate that BSND mutations with different functional consequences are the basis for either syndromic or nonsyndromic deafness.

A comparison of the effects of ivermectin and moxidectin on the nematode Caenorhabditis elegans

The avermectins and the milbemycins are structurally related classes of 16-membered macrocyclic lactones (ML) that have a broad spectrum of activity. Most studies on the mode of action of ML have used the avermectin, ivermectin (IVM). IVM activates glutamate-gated chloride channels that contain alpha-type subunits, resulting in a hyperpolarization of the neuronal membrane, leading to a flaccid paralysis. IVM kills Caenorhabditis elegans at therapeutic concentrations, making it a useful model to examine mechanisms of IVM toxicity and resistance. There have been suggestions that the milbemycins may exert effects that are different from the avermectins, however this hypothesis has been challenged. Using IVM and the milbemycin, moxidectin (MOX), we demonstrate that while the two drugs have some similar effects on C. elegans, there are also some differences in worm response. Following exogenous exposure to a gradient of IVM and MOX, ranging from 0 to 5000 nM, quantitative and qualitative differences in response to the two anthelmintic drugs were observed in the pharyngeal pump rate, larval development and motility of wild-type and glutamate-gated chloride channel (GluCl) subunit knockout strains of C. elegans. After exposure to equimolar drug concentrations, differences between the anthelmintic effects were observed in the motility phenotype in the wild-type, GluCl subunit knockout strains and multi-gene knockout strain of C. elegans that exhibits a marked reduction in IVM sensitivity; and transcription profiles of genes coding for GluCl subunits in both the wild-type and glc-2 knockout strain. The glc-2 deletion strain showed increased motility in response to 2.5 nM MOX in the first 1.5 h of exposure, compared with wild-type nematodes, whereas this strain showed little change in motility in response to IVM. The pharyngeal pump rate in the glc-2 deletion strain was sensitive to equimolar concentrations of IVM and MOX. The triple avr-14/ avr-15/ glc-1 knockout caused a loss of initial stimulation of motility seen in the wild-type, by 2.5 nM IVM, to a reduction in motility, whereas the response to MOX was little changed between this triple knockout strain and wild-type C. elegans. The results suggest that there are significant differences in the response of C. elegans to IVM and MOX. The product of the glc- 2 gene may play a role in sensitivity to MOX, but not to IVM, while the products of avr-14, avr-15 and glc-1 may be important for the effects of IVM, but less so for MOX.

A dopamine-gated ion channel (HcGGR3*) from Haemonchus contortus is expressed in the cervical papillae and is associated with macrocyclic lactone resistance

Haemonchus contortus, a parasitic nematode of great economic importance, is a major challenge for the livestock industries. The parasite is controlled by nematocidal drugs, several of which target ligand-gated chloride channels (LGCCs). In addition, drug resistance has become a major problem in controlling this parasite and other trichostrongylids. Therefore, identification of new drug targets may assist in the discovery of novel drugs which is essential for maintaining the level of parasite control required in modern agriculture. We have isolated a novel LGCC gene, which has been named HcGGR3. Protein sequence analysis indicates that this channel is anion selective and possesses all the signature motifs of a chloride channel subunit. Analysis of the cDNA sequence shows putative microRNA recognition sites which could be important in relation to developmental expression of this subunit. qRT-PCR analysis of HcGGR3 shows that it is differentially expressed among the various life stages and the rank order of expression was eggs > adult female > larvae > adult male. Apart from this, HcGGR3 is significantly down regulated in macrocyclic lactone selected laboratory strains of H. contortus. We also found a single nucleotide polymorphism in the 3′ UTR that appears to be associated with macrocyclic lactone selection. Immunolocalization of this subunit in adult worms has revealed some differences in the expression patterns between males and females. In females, the localization is distinctly punctate around the cervical papillae, suggesting a possible role in mechanosensation. In males, expression was observed around the cervical papillae and possibly some sheath cells. Electrophysiological analysis of this protein expressed in Xenopus laevis oocytes revealed that it forms a homomeric channel that responds primarily to dopamine.

Disease-Causing Dysfunctions of Barttin in Bartter Syndrome Type IV

Bartter syndrome type IV is an inherited human condition characterized by severe renal salt wasting and sensorineural deafness. The causal gene, BSND, encodes barttin, an accessory subunit of chloride channels located in the kidney and inner ear. Barttin modulates the stability, cell surface localization, and function of ClC-K channels; distinct mutations cause phenotypes of varying severity. For definition of the molecular basis of this diversity, the functional consequences of six disease-causing mutations (R8L, R8W, G10S, Q32X, G47R, and E88X) on ClC-K channel properties were studied by heterologous expression in renal cell lines, electrophysiology, confocal imaging, and biochemical analysis. Three missense mutations (R8L, R8W, and G10S) eliminated the function of ClC-K/barttin channels but did not prevent the insertion of the channels into the surface membrane. Another mutant that produces a mild renal phenotype (G47R) was capable of performing all functions of wild-type barttin but bound to ClC-K channels less effectively. The nonsense mutation E88X affected epithelial sorting, leading to equal amounts of barttin inserting into the basolateral and apical membranes, contrasting with the preferential apical insertion of wild-type barttin. Last, the nonsense mutation Q32X allowed barttin to associate with ClC-K channels but prevented surface membrane insertion and channel activation. These results demonstrate that Bartter syndrome type IV can be caused by various derangements in the function of barttin, likely contributing to the diversity of observed phenotypes.

Expression Cloning of TMEM16A as a Calcium-Activated Chloride Channel Subunit

Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.

Ivermectin Interaction with Transmembrane Helices Reveals Widespread Rearrangements during Opening of P2X Receptor Channels

P2X receptors are trimeric cation channels that open in response to binding of extracellular ATP. Each subunit contains a large extracellular ligand binding domain and two flanking transmembrane (TM) helices that form the pore, but the extent of gating motions of the TM helices is unclear. We probed these motions using ivermectin (IVM), a macrocyclic lactone that stabilizes the open state of P2X(4) receptor channels. We find that IVM partitions into lipid membranes and that transfer of the TM regions of P2X(4) receptors is sufficient to convey sensitivity to the lactone, suggesting that IVM interacts most favorably with the open conformation of the two TM helices at the protein-lipid interface. Scanning mutagenesis of the two TMs identifies residues that change environment between closed and open states, and substitutions at a subset of these positions weaken IVM binding. The emerging patterns point to widespread rearrangements of the TM helices during opening of P2X receptor channels.

Thermosensitive TRPV Channel Subunits Coassemble into Heteromeric Channels with Intermediate Conductance and Gating Properties

Heat-sensitive transient receptor potential (TRP) channels (TRPV1–4) form the major cellular sensors for detecting temperature increases. Homomeric channels formed by thermosensitive TRPV subunits exhibit distinct temperature thresholds. While these subunits do share significant sequence similarity, whether they can coassemble into heteromeric channels has been controversial. In the present study we investigated the coassembly of TRPV subunits using both spectroscopy-based fluorescence resonance energy transfer (FRET) and single-channel recordings. Fluorescent protein–tagged TRPV subunits were coexpressed in HEK 293 cells; FRET between different subunits was measured as an indication of the formation of heteromeric channels. We observed strong FRET when fluorescence signals were collected selectively from the plasma membrane using a “spectra FRET” approach but much weaker or no FRET from intracellular fluorescence. In addition, no FRET was detected when TRPV subunits were coexpressed with members of the TRPM subfamily or CLC-0 chloride channel subunits. These results indicate that a substantial fraction of TRP channels in the plasma membrane of cotransfected cells were heteromeric. Single-channel recordings confirmed the existence of multiple heteromeric channel forms. Interestingly, heteromeric TRPV channels exhibit intermediate conductance levels and gating kinetic properties. As these subunits coexpress both in sensory neurons and in other tissues, including heart and brain, coassembly between TRPV subunits may contribute to greater functional diversity.

The cys-loop ligand-gated ion channel gene superfamily of the red flour beetle, Tribolium castaneum

BackgroundMembers of the cys-loop ligand-gated ion channel (cys-loop LGIC) superfamily mediate chemical neurotransmission and are studied extensively as potential targets of drugs used to treat neurological disorders such as Alzheimer's disease. Insect cys-loop LGICs are also of interest as they are targets of highly successful insecticides. The red flour beetle, Tribolium castaneum, is a major pest of stored agricultural products and is also an important model organism for studying development.ResultsAs part of the T. castaneum genome sequencing effort, we have characterized the beetle cys-loop LGIC superfamily which is the third insect superfamily to be described after those of Drosophila melanogaster and Apis mellifera, and also the largest consisting of 24 genes. As with Drosophila and Apis, Tribolium possesses ion channels gated by acetylcholine, γ-amino butyric acid (GABA), glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride channel subunit (pHCl), CG8916 and CG12344. Similar to Drosophila and Apis, Tribolium cys-loop LGIC diversity is broadened by alternative splicing although the beetle orthologs of RDL and GluCl possess more variants of exon 3. Also, RNA A-to-I editing was observed in two Tribolium nicotinic acetylcholine receptor subunits, Tcasα6 and Tcasβ1. Editing in Tcasα6 is evolutionarily conserved with D. melanogaster, A. mellifera and Heliothis virescens, whereas Tcasβ1 is edited at a site so far only observed in the beetle.ConclusionOur findings reveal that in diverse insect species the cys-loop LGIC superfamily has remained compact with only minor changes in gene numbers. However, alternative splicing, RNA editing and the presence of divergent subunits broadens the cys-loop LGIC proteome and generates species-specific receptor isoforms. These findings on Tribolium castaneum enhance our understanding of cys-loop LGIC functional genomics and provide a useful basis for the development of improved insecticides that target an important agricultural pest.

Histamine and Its Receptors Modulate Temperature-Preference Behaviors inDrosophila

Temperature profoundly influences various life phenomena, and most animals have developed mechanisms to respond properly to environmental temperature fluctuations. To identify genes involved in sensing ambient temperature and in responding to its change, >27,000 independent P-element insertion mutants of Drosophila were screened. As a result, we found that defects in the genes encoding for proteins involved in histamine signaling [histidine decarboxylase (hdc), histamine-gated chloride channel subunit 1 (hisCl1), ora transientless (ort)] cause abnormal temperature preferences. The abnormal preferences shown in these mutants were restored by genetic and pharmacological rescue and could be reproduced in wild type using the histamine receptor inhibitors cimetidine and hydroxyzine. Spatial expression of these genes was observed in various brain regions including pars intercerebralis, fan-shaped body, and circadian clock neurons but not in dTRPA1-expressing neurons, an essential element for thermotaxis. We also found that the histaminergic mutants showed reduced tolerance for high temperature and enhanced tolerance for cold temperature. Together, these results suggest that histamine signaling may have important roles in modulating temperature preference and in controlling tolerance of low and high temperature.

Insect GABA Receptors: Splicing, Editing, and Targeting by Antiparasitics and Insecticides

Ionotropic GABA receptors are abundant in both vertebrate and invertebrate nervous systems, where they mediate rapid, mostly inhibitory synaptic transmission. A GABA-gated chloride channel subunit from Drosophila melanogaster [Resistant to Dieldrin (RDL)] has been cloned, functionally expressed, and found to exhibit many aspects of the pharmacology of native, bicuculline-insensitive insect GABA receptors. RDL is the target of the commercially important insecticide fipronil. A point mutation in the channel-lining region of the RDL molecule is known to underlie most cases of resistance to insecticides acting on GABA receptors. RDL is widely distributed throughout the insect nervous system, but the subunit composition of RDL-containing in native receptors is unknown. It is possible that in some instances, RDL coexpresses with glutamate-gated chloride channel subunits. Other ionotropic receptor subunits (LCCH3 and GRD) form GABA-gated cation channels when heterologously expressed. Interest in RDL as a model ligandgated anion channel has been enhanced by the recent discovery of pre-mRNA A-to-I editing, which, together with alternative splicing, adds to the functional diversity of this GABA receptor subunit.

Is anthelmintic resistance a concern for heartworm control?: What can we learn from the human filariasis control programs?

Heartworm prophylaxis is currently largely dependent on the ability of avermectins and milbemycins to arrest the development of third and fourth stages of Dirofilaria immitis for prolonged periods, without producing adulticidal effects. Major control programs, dependent on the activity of ivermectin, are being implemented for human onchocerciasis and lymphatic filariasis. The avermectins and milbemycins act on glutamate-gated and γ-aminobutyrate-gated chloride channel subunit proteins in nematodes. Ivermectin resistance has been widely described in trichostrongylid nematodes of ruminants. There is evidence that when ivermectin resistance occurs in nematodes, there may be selection on some, but not all of the genes that code for ligand-gated chloride channel subunit proteins as well as on some ABC-transporter genes, whose products may be involved in regulating macrocyclic lactone drug concentrations at receptors, and on some structural protein genes of amphidial neurones. Although ivermectin resistance has not been reported in filarial nematodes, there have recently been reports of suboptimal responses to ivermectin in Onchocerca volvulus. Evidence has been found of ivermectin selection on at least ABC-transporter genes and some neuronal structural protein genes in O. volvulus. To date, there is no evidence of avermectin/milbemycin resistance in D. immitis, also a filarial nematode. Chemotherapy against trichostrongylids of animals, human filariae, and D. immitis, relies on avermectins or milbemycins. However, control involves targeting different stages or processes in the nematode life cycles, different control strategies, different proportions of the nematode population in refugia, and different drug dosage rates. Consideration of the proportion of the D. immitis population normally in refugia, the life cycle stage targeted, and the anthelmintic dosages used suggest that it is unlikely that significant avermectin/milbemycin resistance will be selected in D. immitis with current treatment strategies.