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

Li, Ping and Slimko, Eric M. and Lester, Henry A. (2002) Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel. FEBS Letters, 528 (1-3). pp. 77-82. ISSN 0014-5793. https://resolver.caltech.edu/CaltechAUTHORS:20200612-113333592

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Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/s0014-5793(02)03245-3DOIArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2002 by the Biophysical Society. Submitted September 24, 2001, and accepted for publication February 13, 2002. We thank Kira Kostenko and Carrie Shilyansky for help with the oocytes and members of our research group for helpful discussion. This research was supported by a grant from the National Institutes of Health (NS‐11756) and by the William T. Gimbel Discovery Fund in Neuroscience.
Funders:
Funding AgencyGrant Number
NIHNS‐11756
William T. Gimbel Discovery FundUNSPECIFIED
Subject Keywords:Ion channel; Electrical silencing
Issue or Number:1-3
Record Number:CaltechAUTHORS:20200612-113333592
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200612-113333592
Official Citation:Li, Ping, Slimko, Eric M and Lester, Henry A( 2002), Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel, FEBS Letters, 528, doi: 10.1016/S0014-5793(02)03245-3
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103868
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:12 Jun 2020 19:23
Last Modified:12 Jun 2020 19:23

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