High-affinity ivermectin binding to recombinant subunits of the Haemonchus contortus glutamate-gated chloride channel.

Glutamate-gated chloride channels (GluCls) are targets for the avermectin anthelmintics. A family of five GluCl subunit genes encoding seven subunits has been identified in Caenorhabditis elegans. We have previously shown that two orthologous genes in the parasite, Haemonchus contortus, encode three GluCl subunits (HcGluClbeta, Hcgbr-2A and Hcgbr-2B) with high amino-acid identity (>80%) to their C. elegans counterparts. We amplified and cloned a further subunit cDNA, HcGluClalpha, from H. contortus eggs. Sequence comparisons suggested that this subunit was closely related to, but not orthologous with, the C. elegans GluClalpha1, alpha2 or alpha3/GBR-2 subunits ( approximately 55% amino-acid identity). The HcGluClalpha cDNA from an ivermectin-resistant isolate contained no coding changes from the wild-type. All of the known H. contortus GluCl cDNA clones were subcloned into the expression vector pcDNA3.1 and transiently expressed in COS-7 cells. As predicted by functional data from the C. elegans orthologues, the Hcgbr-2A and HcGluClbeta subunits failed to bind [3H]ivermectin. The Hcgbr-2B and HcGluClalpha subunits bound [3H]ivermectin with high affinity; the K(d) values were 70+/-16 and 26+/-12 pM, respectively. This binding was inhibited by a variety of avermectins, though cold ivermectin was the most potent inhibitor of [3H] ivermectin binding. Picrotoxin, fipronil, glutamate and GABA all failed to compete for ivermectin binding to either subunit. The affinity of [3H]ivermectin binding to H. contortus L3 P2 larval membrane preparations was re-examined and found to be 70+/-7 pM. The properties of orthologous GluCl subunits are likely to be conserved across species, but the repertoire and relative importance of those subunits may vary.

Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2.

Remarkable progress has been made recently in identifying a new gene family related to the capsaicin (vanilloid) receptor, VR1. Using a combination of in silico analysis of expressed sequence tag (EST) databases and conventional molecular cloning, we have isolated a novel vanilloid-like receptor, which we call VRL-2, from human kidney. The translated gene shares 46% and 43% identity with VR1 and VRL-1, respectively, and maps to chromosome 12q23-24.1, a locus associated with bipolar affective disorder. VRL-2 mRNA was most strongly expressed in the trachea, kidney, and salivary gland. An affinity-purified antibody against a peptide incorporating the COOH terminal of the receptor localized VRL-2 immunolabel in the distal tubules of the kidney, the epithelial linings of both trachea and lung airways, serous cells of submucosal glands, and mononuclear cells. Unlike VR1 and VRL-1, VRL-2 was not detected in cell bodies of dorsal root ganglia (DRG) or sensory nerve fibers. However, VRL-2 was found on sympathetic and parasympathetic nerve fibers, such as those innervating the arrector pili smooth muscle in skin, sweat glands, intestine, and blood vessels. At least four vanilloid receptor-like genes exist, the newest member, VRL-2 is found in airway and kidney epithelia and in the autonomic nervous system.

L-glutamate binding sites of parasitic nematodes: an association with ivermectin resistance?

Nematode membrane preparations contain high amounts of low-affinity specific L-glutamate binding sites. The numbers of these sites were increased in 2 isolates, one field-derived and the other laboratory-derived, of ivermectin-resistant Haemonchus contortus and a field isolate of ivermectin-resistant Telodorsagia circumcincta, when compared to control, drug-sensitive isolates. Specific [3H]ivermectin binding to these membrane preparations showed no differences between ivermectin-sensitive and resistant isolates and the number of ivermectin binding sites was approximately 100-fold less than the number of L-glutamate binding sites. Kinetic analysis of L-glutamate binding suggested the presence of at least 2 classes of binding site. L-Glutamate binding was blocked by ibotenic acid, kynurenic acid and beta-hydroxyaspartate, but not by ivermectin, argiopine, kainate, quisqualate or NMDA. Competition assays with ibotenic acid suggested that there were 2 distinct populations of glutamate binding sites and that the site with the lower affinity for ibotenate was upregulated in the ivermectin-resistant nematodes. In the field isolate of resistant H. contortus we found no coding changes in the cDNAs encoding glutamate-gated chloride channel subunits HG2, HG3 and HG4, nor were any changes in channel expression detected using subunit-specific antibodies. The low-affinity binding site is unlikely to be associated with the ivermectin receptor in these nematodes.

Cloning and localisation of an avermectin receptor-related subunit from Haemonchus contortus.

Ivermectin is believed to exert its anthelminthic effects by binding to glutamate-gated chloride channels (Glu-Cl) and several cDNAs encoding subunits of Glu-Cl have been cloned from Caenorhabditis elegans. We report the cloning of cDNAs encoding a putative Glu-Cl subunit (HG4) from the parasite Haemonchus contortus. The HG4 cDNAs were isolated using RT-PCR and the sequence of the predicted polypeptide has 82% amino-acid identity with the C. elegans Glu-Cl beta subunit. Individual HG4 cDNAs showed up to 4% sequence variation at the nucleotide level, but the vast majority of these polymorphisms were translationally silent. A synthetic peptide corresponding to sequence near the N-terminus of the mature polypeptide was used to raise an antiserum that recognised the N-terminal domain of HG4 expressed in E. coli. Affinity purified antibodies reacted with motor neuron commissures in immuno-localisation studies: these commissures were limited to the anterior portion of the worms, from a region level with the nerve ring to just anterior of the vulva. Some possible nerve cord staining was also observed, but no expression of HG4 on pharyngeal muscle could be detected.

The unfolded-protein-response element discriminates misfolding induced by different mutant pro-sequences of yeast carboxypeptidase Y.

The C-terminal region of the chaperone-like pro-sequence (py) of yeast carboxypeptidase Y (CPY) is suggested to be crucial for the folding of mature CPY. In order to study the influence of hydrophobic residues in this domain, a set of mutations have been introduced in py. Unexpectedly, only small amounts of CPY precursors are expressed when Leu108, at the C-terminal end of py, is substituted for polar residues Lys, Arg or Asp. In contrast, substitution with hydrophobic residues Val, Ile or Ala permit normal expression. Interestingly, the poorly expressed molecules are coreglycosylated, implying that they have failed to leave the endoplasmic reticulum (ER). The ER-retained molecules caused an induction in the levels of BiP, signifying that polar substitutions at position 108 of pre-py-CPY induce misfolding. Quite surprisingly, a reporter gene, linked to concatamerized unfolded-protein-response elements, reveals that py-mediated misfolding of CPY is not really identical in all mutants. This shows that a simple transcriptional assay can assess the subtleties of pro-sequence mediated protein folding in an eukaryotic cell.