RESUMEN
Nematode glutamate-gated chloride channels are targets of the macrocyclic lactones, the most important group of anthelmintics available. In Xenopus laevis oocytes, channels formed by the GluClalpha3B subunit from the parasite Haemonchus contortus were more sensitive to l-glutamate (EC(50) = 27.6 +/- 2.7 microM) than those formed by the homologous subunit from Caenorhabditis elegans (EC(50) = 2.2 +/- 0.12 mM). Ibotenate was a partial agonist (EC(50) = 87.7 +/- 3.5 microM). The H. contortus channels responded to low concentrations of ivermectin (estimated EC(50) = approximately 0.1 +/- 1.0 nM), opening slowly and irreversibly in a highly cooperative manner: the rate of channel opening was concentration-dependent. Responses to glutamate and ivermectin were inhibited by picrotoxinin and fipronil. Mutating an N-terminal domain amino acid, leucine 256, to phenylalanine increased the EC(50) for l-glutamate to 92.2 +/- 3.5 microM, and reduced the Hill number from 1.89 +/- 0.35 to 1.09 +/- 0.16. It increased the K(d) for radiolabeled ivermectin binding from 0.35 +/- 0.1 to 2.26 +/- 0.78 nM. Two other mutations (E114G and V235A) had no effect on l-glutamate activation or ivermectin binding: one (T300S) produced no detectable channel activity, but ivermectin binding was similar to wild-type. The substitution of any aromatic amino acid for Leu256 had similar effects in the radioligand binding assay. Molecular modeling studies suggested that the GluCl subunits have a fold similar to that of other Cys-loop ligand-gated ion channels and that amino acid 256 was unlikely to play a direct role in ligand binding but may be involved in mediating the allosteric properties of the receptor.
Asunto(s)
Antihelmínticos/farmacología , Agonistas de los Canales de Cloruro , Haemonchus/metabolismo , Ivermectina/farmacología , Animales , Células COS , Canales de Cloruro/genética , Chlorocebus aethiops , Resistencia a Medicamentos , Femenino , Ácido Glutámico/farmacología , Activación del Canal Iónico , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Conformación Proteica , Subunidades de Proteína/fisiología , Ensayo de Unión Radioligante , Xenopus laevisRESUMEN
Ionic transport (for applications in nanofluidics or membranes) and "ionic diode" phenomena in a zeolitic imidazolate framework (ZIF-8) are investigated by directly growing the framework from aqueous Zn(2+) and 2-methylimidazole as an "asymmetric plug" into a 20 µm diameter pore in a ca. 6 µm thin poly-ethylene-terephthalate (PET) film.