Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel

FEBS Letters

Slimko, Eric M²  Li, Ping¹  Lester, Henry A¹ 

[1] Division of Biology, M/C 156-29, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA 91125, USA;Computation and Neural Systems Program, California Institute of Technology, Pasadena, CA 91125, USA

关键词: Ion channel;    Electrical silencing;   

DOI  :  10.1016/S0014-5793(02)03245-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 β-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 α. For GluCl αβ(Y182F), the EC₅₀ 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 α, β and β(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.

【 授权许可】

Unknown