Binding characteristics of emamectin benzoate to the putative glutamate-gated chloride channels of Lepeophtheirus salmonis.

摘要

The objective of the present study was to analyze some of the binding characteristics of emamectin benzoate (EMB) to the glutamate-gated chloride channels (GluCl) of the salmon louse, Lepeophtheirus salmonis. This drug is used to control sea lice infestation in farmed Atlantic salmon.;Membranes from both sea lice and the CHSE-214 cell line were used in a binding assay to determine binding characteristics. The putative gene encoding the GluCl of L. salmonis was transfected into the cell line. The assay involved incubating the membranes at concentrations of 0, 0.1, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 nM of [3H]EMB for a minimum of two hours at 15°C and then terminating the reaction by filtration through glass fibre filter paper. The radioactivity of the protein retained on the filter paper was measured using a liquid scintillation counter. The calculated dissociation constant (KD) of EMB on membranes extracted from the sea lice was 1.377 +/- 0.205 nM. No binding was detected in the cell line membranes, and no GluClalpha protein was detected by Western blotting, indicating a potential inability of the CHSE cells to express GluCl. Binding assays were repeated on the membranes extracted from sea lice at 5°C and 20°C. The KD calculated at these temperatures was 1.921 +/- 0.715 nM and 1.578 +/- 0.145 nM respectively. There was no significant change in the KD, but there was an increase in nonspecific binding at 5°C which resulted in high variability. The maximum number of receptors bound (BMAX) was 9.049 +/- 0.464 pmol/mg of protein at 15°C, 8.588 +/- 0.268 pmol/mg of protein at 20°C, and 9.642 +/- 0.822 pmol/mg at 5°C.;One of the potential mechanisms of EMB resistance development in sea lice involves a reduction or loss of drug affinity for the GluCl receptor due to a mutation which changes the conformation of the avermectin binding site. This study confirmed the presence of one or more binding sites in the membrane of the sea lice and provided a measurement of the level of affinity. This site may correspond to a GluClalpha receptor. The results indicate that temperature has no significant effect on the affinity of the drug to the substrate. These data, coupled with a cell line expressing the GluCl receptor, can be used to develop a model to determine whether or not affinity is a cause for loss of sensitivity in resistant organisms.