Chloride Channel Subunit Research Articles

The Utility of BSND Immunohistochemistry in the Differential Diagnosis of Oncocytic and Warthin-like Mucoepidermoid Carcinoma of Salivary Gland.

BSND is a chloride channel subunit that is expressed in the normal salivary gland. We aimed to validate the utility of BSND immunohistochemistry in the differential diagnosis of oncocytic salivary gland neoplasms. BSND immunohistochemistry was performed in a retrospective cohort of 93 salivary gland lesions, enriched with tumors with oncocytic features and histologic variants of mucoepidermoid carcinoma (MEC). All oncocytomas (n = 18) showed diffuse membranous BSND immunopositivity. Warthin tumors (n = 18) were also positive for BSND, but the staining pattern was patchy cytoplasmic and membranous in 10-25% of tumor cells. Using a threshold of 10% BSND-positive cells, all Warthin tumors were positive, while none of Warthin-like MECs or non-MEC salivary tumors were positive. Applying the same 10% positivity criterion, two oncocytic MECs were positive for BSND. The percentage of BSND staining in oncocytic MECs was up to 20%. In contrast, BSND was diffusely positive in oncocytomas with a median percentage of positivity of 95% (range: 40 - 100%). Therefore, a higher threshold of > 20% BSND-positive cells may be considered when differentiating between oncocytoma and oncocytic MEC. BSND immunohistochemistry is a potentially useful diagnostic marker for salivary gland neoplasms, especially oncocytic and Warthin-like MECs. A threshold of ≥ 10% positivity can differentiate Warthin tumors from Warthin-like MECs, whereas > 20% positivity can be effective for separating oncocytomas from oncocytic MECs.

The Evolution and Mechanisms of Multiple-Insecticide Resistance in Rice Stem Borer, Chilo suppressalis Walker (Lepidoptera: Crambidae).

The emergence of insecticide resistance in the rice stem borer, Chilo suppressalis, is a growing threat to the sustainable control of this important insect crop pest. Thus, monitoring of C. suppressalis populations for insecticide resistance and characterization of the underlying genetic mechanisms is essential to inform rational control decisions and the development of resistance management strategies. Here, we monitored 126 C. suppressalis field populations from China for resistance evolution to four major insecticides: 53 for chlorantraniliprole, 50 for abamectin, 74 for triazophos, and 76 for spinetoram. Moderate to high levels of resistance were observed to all four insecticides. Investigation of the underlying resistance mechanisms revealed multiple mutations in the ryanodine receptor (RyR) and acetylcholinesterase 1 (AChE1), leading to target-site resistance to chlorantraniliprole and triazophos, respectively. In contrast, the absence of mutations in the glutamate-gated chloride channel (GluCl) and α6 nicotinic acetylcholine receptor (nAChR α6) subunit suggested that nontarget site mechanisms contribute to the multiple-insecticide resistance phenotypes observed in C. suppressalis. In this regard, we revealed overexpression of the uridine 5'-diphospho-glycosyltransferase UGT33AF1 and cytochrome P450 CYP6AB45 in C. suppressalis field populations. Functional characterization using transgenic Drosophila demonstrated that UGT33AF1 confers resistance against multiple insecticides in vivo, whereas CYP6AB45 does not appear to contribute to resistance. Collectively, our findings reveal the current status of resistance of C. suppressalis to insecticides in China and uncover a diverse profile of resistance mechanisms in this species. These findings provide a foundation for the development of sustainable strategies to effectively manage and control this pest.

LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions

Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl- efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na+/K+/2Cl- cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.

Identification and characterization of striking multiple-insecticide resistance in a Tetranychus urticae field population from Greece.

Tetranychus urticae is a notorious crop pest with a worldwide distribution that has developed resistance to a wide range of acaricides. Here, we investigated the resistance levels of a T. urticae population collected from an ornamental greenhouse in Peloponnese, Greece, and analyzed its resistance mechanisms at the molecular level. Toxicological assays showed resistance against compounds with different modes of action, with resistance ratios of: 89-fold for abamectin; > 1000-fold for clofentezine; > 5000-fold for etoxazole; 27-fold for fenpyroximate and pyridaben; 20- and 36-fold for spirodiclofen and spirotetramat, respectively; and 116- and > 500-fold for cyenopyrafen and cyflumetofen, respectively. Bioassays with synergists indicated the involvement of detoxification enzymes in resistance to abamectin, but not to cyflumetofen and spirodiclofen. RNA sequencing (RNA-seq) analysis showed significant over-expression of several genes encoding detoxification enzymes such as cytochrome P450 monooxygenases and UDP-glycosyltransferases, which have been previously associated with acaricide resistance. Known target-site resistance mutations were identified in acetyl-choline esterase, chitin synthase 1 and NDUFS7/psst, but putative novel resistance mutations were also discovered in targets such as glutamate-gated chloride channel subunit 3. Interestingly, target-site resistance mutations against pyrethroids or bifenazate were not identified, possibly indicating a recent reduced selection pressure in Greece, as well as a possible opportunity to rotate these chemistries. We identified and characterized a striking case of multiple acaricide resistance in a field population of T. urticae. Exceptionally strong resistance phenotypes, with accumulation of multiple resistance mutations and over-expression of P450s and other detoxification genes in the same field population are reported.

DHHC7-mediated palmitoylation of the accessory protein barttin critically regulates the functions of ClC-K chloride channels

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7-/- mice. Although palmitoylation of barttin in kidneys of Zdhhc7-/- animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7-/- mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

In silico identification and assessment of insecticide target sites in the genome of the small hive beetle, Aethina tumida

BackgroundThe small hive beetle, Aethina tumida, is a rapidly emerging global pest of honey bee colonies. Small hive beetle infestation can be extremely destructive, which may cause honey bees to abscond and render colony infrastructure unusable. Due to the impacts small hive beetles have on honey bees, a wide variety of physical, cultural, and chemical control measures have been implemented to manage small hive beetle infestations. The use of insecticides to control small hive beetle populations is an emerging management tactic. Currently, very little genomic information exists on insecticide target sites in the small hive beetle. Therefore, the objective of this study is to utilize focused in silico comparative genomics approaches to identify and assess the potential insecticide sensitivity of the major insecticide target sites in the small hive beetle genome.ResultsNo previously described resistance mutations were identified in any orthologs of insecticide target sites. Alternative exon use and A-to-I RNA editing were absent in AtumSC1. The ryanodine receptor in small hive beetle (Atum_Ryr) was highly conserved and no previously described resistance mutations were identified. A total of 12 nAChR subunits were identified with similar alternative exon use in other insects. Alternative exon use and critical structural features of the GABA-gated chloride channel subunits (Atum_RDL, Atum_GRD, and Atum_LCCH3) were conserved. Five splice variants were found for the glutamate-gated chloride channel subunit. Exon 3c of Atum_GluCl may be a beetle-specific alternative exon. The co-occurrence of exons 9a and 9b in the pH-sensitive chloride channel (Atum_pHCl) is a unique combination that introduces sites of post-translational modification. The repertoire and alternative exon use for histamine-gated chloride channels (Atum-HisCl), octopamine (Atum_OctR) and tyramine receptors (Atum_TAR) were conserved.ConclusionsThe recently published small hive beetle genome likely serves as a reference for insecticide-susceptible versions of insecticide target sites. These comparative in silico studies are the first step in discovering targets that can be exploited for small hive beetle-specific control as well as tracking changes in the frequency of resistance alleles as part of a resistance monitoring program. Comparative toxicity alongside honey bees is required to verify these in silico predictions.

Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs.

Barttin is an accessory subunit of ClC-K chloride channels expressed in the kidney and the inner ear. Main functions of ClC-K/barttin channels are the generation of the cortico-medullary osmotic gradients in the kidney and the endocochlear potential in the inner ear. Mutations in the gene encoding barttin, BSND, result in impaired urinary concentration and sensory deafness. Barttin is predicted to be a two helical integral membrane protein that directly interacts with its ion channel in the membrane bilayer where it stabilizes the channel complex, promotes its incorporation into the surface membrane and leads to channel activation. It therefore is an attractive target to address fundamental questions of intermolecular communication within the membrane. However, so far inherent challenges in protein expression and stabilization prevented comprehensive in vitro studies and structural characterization. Here we demonstrate that cell-free expression enables production of sufficient quantities of an isotope-labeled barttin variant (I72X Barttin, capable to promote surface membrane insertion and channel activation) for NMR-based structural studies. Additionally, we established purification protocols as well as reconstitution strategies in detergent micelles and phospholipid bilayer nanodiscs. Stability, folding, and NMR data quality are reported as well as a suitable assignment strategy, paving the way to its structural characterization.

Abnormal Scn1b and Fxyd1 gene expression in the pulled-through ganglionic colon may influence functional outcome in patients with Hirschsprung's disease.

Smooth muscle cells are electrically coupled to ICC and PDGFRα+ cells, to regulate smooth muscle contraction. Recent studies have reported that the voltage-gated sodium channel type 1β (Scn1b), and the chloride channel subunit, Fxyd1, are highly expressed by both ICC and PDGFRα+ cells in the mouse colon. We designed this study to investigate the expression of the Scn1b and Fxyd1 genes in the normal human colon and in HSCR. HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify Scn1b and Fxyd1 gene expression, and immunolabelling of Scn1b and Fxyd1 proteins were visualized using confocal microscopy. qRT-PCR analysis revealed significant downregulation of Scn1b and Fxyd1 genes in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed a reduction in Scn1b and Fxyd1 protein expression in both aganglionic and ganglionic HSCR colon compared to controls. Scn1b and Fxyd1 expression was significantly downregulated in HSCR colon. These results add to mounting evidence suggesting that the pulled-through ganglionic segment of bowel in these patients is abnormal, despite the presence of ganglion cells.

Investigating the function and possible biological role of an acetylcholine-gated chloride channel subunit (ACC-1) from the parasitic nematode Haemonchus contortus

The cys-loop superfamily of ligand-gated ion channels are well recognized as important drug targets for many invertebrate specific compounds. With the rise in resistance seen worldwide to existing anthelmintics, novel drug targets must be identified so new treatments can be developed. The acetylcholine-gated chloride channel (ACC) family is a unique family of cholinergic receptors that have been shown, using Caenorhabditis elegans as a model, to have potential as anti-parasitic drug targets. However, there is little known about the function of these receptors in parasitic nematodes. Here, we have identified an acc gene (hco-acc-1) from the sheep parasitic nematode Haemonchus contortus. While similar in sequence to the previously characterized C. elegans ACC-1 receptor, Hco-ACC-1 does not form a functional homomeric channel in Xenopus oocytes. Instead, co-expression of Hco-ACC-1 with a previously characterized subunit Hco-ACC-2 produced a functional heteromeric channel which was 3x more sensitive to acetylcholine compared to the Hco-ACC-2 homomeric channel. We have also found that Hco-ACC-1 can be functionally expressed in C. elegans. Overexpression of both cel-acc-1 and hco-acc-1 in both C. elegans N2 and acc-1 null mutants decreased the time for worms to initiate reversal avoidance to octanol. Moreover, antibodies were generated against the Hco-ACC-1 protein for use in immunolocalization studies. Hco-ACC-1 consistently localized to the anterior half of the pharynx, specifically in pharyngeal muscle tissue in H. contortus. On the other hand, expression of Hco-ACC-1 in C. elegans was restricted to neuronal tissue. Overall, this research has provided new insight into the potential role of ACC receptors in parasitic nematodes.

A G326E substitution in the glutamate-gated chloride channel 3 (GluCl3) of the two-spotted spider mite Tetranychus urticae abolishes the agonistic activity of macrocyclic lactones.

The macrocyclic lactones abamectin and milbemectin are frequently used to control phytophagous mites such as Tetranychus urticae. Consequently, resistance has developed and was genetically linked with substitutions in the glutamate-gated chloride channel (GluCl) subunits TuGluCl1 and TuGluCl3. Here, we functionally validated a G326E substitution in TuGluCl3 by functional expression in Xenopus laevis oocytes followed by two-electrode voltage-clamp electrophysiology. Homomeric wild-type and mutated GluCl3 were successfully expressed. l-glutamic-acid-induced currents exhibited a rapid onset equal in both channels and EC50 for l-glutamic-acid was in the micromolar range (384.2 μm and 292.7 μm, respectively). Abamectin and milbemycin A4 elicited sustained currents in wild-type GluCl3, but the G326E substitution completely abolished the agonistic activity of macrocyclic lactones. A target-site mutation in Tu GluCl3 contributes to avermectin resistance in T. urticae. However, given the multitude of channel genes and the potential additive or synergistic effects of mutations, to what extent mutations determine the often extremely strong resistance phenotype in the field deserves further study. © 2017 Society of Chemical Industry.

Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster.

Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster. Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets.

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 μM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30mM TMEM16A current activates with a monoexponential time course, but above 30mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.

Pharmacological characterization of histamine-gated chloride channels from the housefly Musca domestica

The biogenic amine histamine (HA) is not only the neurotransmitter of photoreceptors but also has important roles in mechanosensory reception, temperature preference, sleep and olfactory processing in insects. Two cDNAs (MdhclA and MdhclB) that encode HA-gated chloride channel subunits (MdHCLA and MdHCLB) were cloned from the housefly Musca domestica. The cRNAs were injected into Xenopus laevis oocytes to examine the functions and pharmacological characteristics of MdHCLA and MdHCLB channels using a two-electrode voltage clamp method. HA was used to activate MdHCLA and MdHCLB channels to evoke inward currents with EC50s of 33.1μM and 6.28μM, respectively. 2-(3-Trifluoromethylphenyl)histamine, an HA H1 receptor agonist, was a partial agonist of MdHCLB receptors with an EC50 of 49.4μM. MdHCLB channels were also activated by γ-aminobutyric acid (GABA) and monoamines, such as octopamine, serotonin (5-HT) and dopamine (DA); 5-HT and DA also acted as competitive antagonists. GABA acted as a full agonist of MdHCLB receptors with an EC50 of 1.11mM. d-Tubocurarine, cimetidine and picrotoxinin were poor inhibitors of HA- and GABA-evoked currents in MdHCLB channels. Our data show that HCLB channels are more sensitive to agonists when compared with HCLA channels. HCLB channels are also affected by antagonists but insusceptible to known insecticides that target GABA- and glutamate-gated chloride channels.

Molecular mechanisms of Tetranychus urticae chemical adaptation in hop fields.

The two-spotted spider mite, Tetranychus urticae Koch is a major pest that feeds on >1,100 plant species. Many perennial crops including hop (Humulus lupulus) are routinely plagued by T. urticae infestations. Hop is a specialty crop in Pacific Northwest states, where 99% of all U.S. hops are produced. To suppress T. urticae, growers often apply various acaricides. Unfortunately T. urticae has been documented to quickly develop resistance to these acaricides which directly cause control failures. Here, we investigated resistance ratios and distribution of multiple resistance-associated mutations in field collected T. urticae samples compared with a susceptible population. Our research revealed that a mutation in the cytochrome b gene (G126S) in 35% tested T. urticae populations and a mutation in the voltage-gated sodium channel gene (F1538I) in 66.7% populations may contribute resistance to bifenazate and bifenthrin, respectively. No mutations were detected in Glutamate-gated chloride channel subunits tested, suggesting target site insensitivity may not be important in our hop T. urticae resistance to abamectin. However, P450-mediated detoxification was observed and is a putative mechanism for abamectin resistance. Molecular mechanisms of T. urticae chemical adaptation in hopyards is imperative new information that will help growers develop effective and sustainable management strategies.

Glutamate-gated chloride channel subunit cDNA sequencing of Cochliomyia hominivorax (Diptera: Calliphoridae): cDNA variants and polymorphisms.

The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel) is one of the major myiasis-causing flies that injures livestock and leads to losses of ~US$ 2.7 billions/year in the Neotropics. Ivermectin (IVM), a macrocyclic lactone (ML), is the most used preventive insecticide for this parasite and targets the glutamate-gated chloride (GLUCLα) channels. Several authors have associated altered GluClα homologues to MLs resistance in invertebrates, although studies about resistance in NWS are limited to other genes. Here, we aimed to characterise the NWS GluClα (ChGluClα) cDNA and to search for alterations associated with IVM resistance in NWS larvae from a bioassay. The open reading frame of the ChGluClα comprised 1,359 bp and encoded a sequence of 452 amino acids. The ChGluClα cDNAs of the bioassay larvae showed different sequences that could be splice variants, which agree with the occurrence of alternative splicing in GluClα homologues. In addition, we found cDNAs with premature stop codons and the K242R SNP, which occurred more frequently in the surviving larvae and was located close to mutation (L256F) involved in ML resistance. Although these alterations were in low frequency, the ChGluClα sequencing will allow further studies to find alterations in the gene of resistant natural populations.

Severe manifestation of Bartter syndrome Type IV caused by a novel insertion mutation in the BSND gene

Bartter syndrome Type IV is a rare subtype of the Bartter syndromes that leads to both severe renal salt wasting and sensorineural deafness. This autosomal recessive disease is caused by mutations in the gene encoding barttin, BSND, an essential subunit of the ClC-K chloride channels expressed in renal and inner ear epithelia. Patients differ in the severity of renal symptoms, which appears to depend on the modification of channel function by the mutant barttin. To date, only a few BSND mutations have been reported, most of which are missense or nonsense mutations. In this study, we report the identification of the first insertion mutation, p.W102Vfs*7, in the BSND gene of a newborn girl with acute clinical symptoms including early-onset chronic renal failure. The results support previous data indicating that mutations that are predicted to abolish barttin expression are associated with a severe phenotype and early onset renal failure.

Pharmacological characterization of TMEM16A currents

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACCinhA01 and T16AinhA01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HEK 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACCinhA01 and T16AinhA01 inhibited TMEM16A currents with IC50 values of 12, 58, 1.7 and 1.5 µM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACCinhA01 and T16AinhA01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACCinhA01 and T16AinhA01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.

Molecular Cloning and Characterization of Novel Glutamate-Gated Chloride Channel Subunits from Schistosoma mansoni

Cys-loop ligand-gated ion channels (LGICs) mediate fast ionotropic neurotransmission. They are proven drug targets in nematodes and arthropods, but are poorly characterized in flatworms. In this study, we characterized the anion-selective, non-acetylcholine-gated Cys-loop LGICs from Schistosoma mansoni. Full-length cDNAs were obtained for SmGluCl-1 (Smp_096480), SmGluCl-2 (Smp_015630) and SmGluCl-3 (Smp_104890). A partial cDNA was retrieved for SmGluCl-4 (Smp_099500/Smp_176730). Phylogenetic analyses suggest that SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 belong to a novel clade of flatworm glutamate-gated chloride channels (GluCl) that includes putative genes from trematodes and cestodes. The flatworm GluCl clade was distinct from the nematode-arthropod and mollusc GluCl clades, and from all GABA receptors. We found no evidence of GABA receptors in S. mansoni. SmGluCl-1, SmGluCl-2 and SmGluCl-3 subunits were characterized by two-electrode voltage clamp (TEVC) in Xenopus oocytes, and shown to encode Cl−-permeable channels gated by glutamate. SmGluCl-2 and SmGluCl-3 produced functional homomers, while SmGluCl-1 formed heteromers with SmGluCl-2. Concentration-response relationships revealed that the sensitivity of SmGluCl receptors to L-glutamate is among the highest reported for GluCl receptors, with EC50 values of 7–26 µM. Chloride selectivity was confirmed by current-voltage (I/V) relationships. SmGluCl receptors are insensitive to 1 µM ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluClα subtype group. SmGluCl receptors are also insensitive to 10 µM meclonazepam, a schistosomicidal benzodiazepine. These results provide the first molecular evidence showing the contribution of GluCl receptors to L-glutamate signaling in S. mansoni, an unprecedented finding in parasitic flatworms. Further work is needed to elucidate the roles of GluCl receptors in schistosomes and to explore their potential as drug targets.

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster.

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala(301) to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala(301) to Ser resistance mutation and Met(360) to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser(301) homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser(301) change into an Ala(301) background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene.

Histamine-HisCl1 Receptor Axis Regulates Wake-Promoting Signals in Drosophila melanogaster

Histamine and its two receptors, histamine-gated chloride channel subunit 1 (HisCl1) and ora transientless (Ort), are known to control photoreception and temperature sensing in Drosophila. However, histamine signaling in the context of neural circuitry for sleep-wake behaviors has not yet been examined in detail. Here, we obtained mutant flies with compromised or enhanced histamine signaling and tested their baseline sleep. Hypomorphic mutations in histidine decarboxylase (HDC), an enzyme catalyzing the conversion from histidine to histamine, caused an increase in sleep duration. Interestingly, hisCl1 mutants but not ort mutants showed long-sleep phenotypes similar to those in hdc mutants. Increased sleep duration in hisCl1 mutants was rescued by overexpressing hisCl1 in circadian pacemaker neurons expressing a neuropeptide pigment dispersing factor (PDF). Consistently, RNA interference (RNAi)-mediated depletion of hisCl1 in PDF neurons was sufficient to mimic hisCl1 mutant phenotypes, suggesting that PDF neurons are crucial for sleep regulation by the histamine-HisCl1 signaling. Finally, either hisCl1 mutation or genetic ablation of PDF neurons dampened wake-promoting effects of elevated histamine signaling via direct histamine administration. Taken together, these data clearly demonstrate that the histamine-HisCl1 receptor axis can activate and maintain the wake state in Drosophila and that wake-activating signals may travel via the PDF neurons.