Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel.
Glutamate-gated chloride (GluCl) channels from invertebrates can be activated by ivermectin (IVM) to produce electrical silencing in mammalian neurons. To improve this GluCl/IVM strategy, we sought to mutate the Caenorhabditis elegans GluCl channels so that they become insensitive to glutamate but retain their sensitivity to IVM. Based on structure-function studies of nicotinic acetylcholine receptor superfamily members, we tested in oocytes 19 point mutants at 16 residues in the beta-subunit likely to be involved in the response to glutamate. Y182F reduces the glutamate response by greater than six-fold, with little change to IVM responses, when coexpressed with wild-type (WT) GluCl alpha. For GluCl alphabeta(Y182F), the EC(50) and Hill coefficient for glutamate are similar to those of WT, indicating that the mutant decreases the efficacy of glutamate, but not the potency. Also, fluorescent proteins (enhanced green fluorescent protein, enhanced yellow fluorescent protein, enhanced cyan fluorescent protein; XFP) were inserted into the M3-M4 loop of the GluCl alpha, beta and beta(Y182F). We found no significant functional difference between these XFP-tagged receptors and WT receptors. The modified GluCl channel, without glutamate sensitivity but with a fluorescent tag, may be more useful in GluCl silencing strategies.
Li
Ping
P
Division of Biology, M/C 156-29, California Institute of Technology, Pasadena 91125, USA.
Slimko
Eric M
EM
Lester
Henry A
HA
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D066298:In Vitro Techniques; D007559:Ivermectin / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D001426:Bacterial Proteins; D029742:Caenorhabditis elegans Proteins; D018118:Chloride Channels; D018076:DNA, Complementary; D008024:Ligands; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C109777:glutamate-gated chloride channels; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D007559:Ivermectin