Bidirectional regulation of thermotaxis by glutamate transmissions in Caenorhabditis elegans

This paper provides a molecular and genetic analysis of the neural circuitry that regulates the migration of Caenorhabditis elegans towards either warmer or colder temperature and reveals an important role of glutamate signalling in this process

Glutamate-Gated Chloride Channels of Haemonchus contortus Restore Drug Sensitivity to Ivermectin Resistant Caenorhabditis elegans

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance

Transient Receptor Potential Vanilloid 1 and Xenobiotics

Over the last couple of years, transient receptor potential vanilloid 1(TRPV1) channels have been a hot topic in ion channel research. Since this research field is still rather new, there is not much known about the working mechanism of TRPV1 and its ligands. Nevertheless, the important physiological role and therapeutic potential are promising. Therefore, extensive research is going on and a lot of natural as well as synthetic compounds are already described. In this review, we briefly give an overview of capsaicin's history and the current knowledge of its working mechanism and physiological role. We discuss the best known plant molecules acting on TRPV1 and highlight the latest discovery in TRPV1 research: animal venoms and toxins acting on TRPV1 channels. In an effort to give the complete image of TRPV1 ligands known today, the most promising synthetic compounds are presented. Finally, we present a novel pharmacophore model describing putative ligand binding domains.status: publishe

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 106 bp 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

GLC-3: a novel fipronil and BIDN-sensitive, but picrotoxinin-insensitive, L-glutamate-gated chloride channel subunit from Caenorhabditis elegans

1. We report the cloning and expression of a novel Caenorhabditis elegans polypeptide, GLC-3, with high sequence identity to previously cloned L-glutamate-gated chloride channel subunits from nematodes and insects. 2. Expression of glc-3 cRNA in Xenopus oocytes resulted in the formation of homo-oligomeric L-glutamate-gated chloride channels with robust and rapidly desensitizing currents, an EC(50) of 1.9±0.03 mM and a Hill coefficient of 1.5±0.1. GABA, glycine, histamine and NMDA all failed to activate the GLC-3 homo-oligomer at concentrations of 1 mM. The anthelminthic, ivermectin, directly and irreversibly activated the L-glutamate-gated channel with an EC(50) of 0.4±0.02 μM. 3. The GLC-3 channels were selective for chloride ions, as shown by the shift in the reversal potential for L-glutamate-gated currents after the reduction of external Cl(−) from 107.6 to 62.5 mM. 4. Picrotoxinin failed to inhibit L-glutamate agonist responses at concentrations up to 1 mM. The polycyclic dinitrile, 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile (BIDN), completely blocked L-glutamate-induced chloride currents recorded from oocytes expressing GLC-3 with an IC(50) of 0.2±0.07 μM. The phenylpyrazole insecticide, fipronil, reversibly inhibited L-glutamate-gated currents recorded from the GLC-3 receptor with an IC(50) of 11.5±0.11 μM. 5. In this study, we detail the unusual antagonist pharmacology of a new GluCl subunit from C. elegans. Unlike all other native and recombinant nematode GluCl reported to date, the GLC-3 receptor is insensitive to picrotoxinin, but is sensitive to two other channel blockers, BIDN and fipronil. Further study of this receptor may provide insights into the molecular basis of non-competitive antagonism by these compounds

Expression of nicotinic acetylcholine receptor subunits from parasitic nematodes in Caenorhabditis elegans

The levamisole-sensitive nicotinic acetylcholine receptor present at nematode neuromuscular junctions is composed of multiple different subunits, with the exact composition varying between species. We tested the ability of two well-conserved nicotinic receptor subunits, UNC-38 and UNC-29, from Haemonchus contortus and Ascaris suum to rescue the levamisole-resistance and locomotion defects of Caenorhabditis elegans strains with null deletion mutations in the unc-38 and unc-29 genes. The parasite cDNAs were cloned downstream of the relevant C. elegans promoters and introduced into the mutant strains via biolistic transformation. The UNC-38 subunit of H. contortus was able to completely rescue both the locomotion defects and levamisole resistance of the null deletion mutant VC2937 (ok2896), but no C. elegans expressing the A. suum UNC-38 could be detected. The H. contortus UNC-29.1 subunit partially rescued the levamisole resistance of a C. elegans null mutation in unc-29 VC1944 (ok2450), but did cause increased motility in a thrashing assay. In contrast, only a single line of worms containing the A. suum UNC-29 subunit showed a partial rescue of levamisole resistance, with no effect on thrashing