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An Engineered Glutamate-gated Chloride (GluCl) Channel for Sensitive, Consistent Neuronal Silencing by Ivermectin

Frazier, Shawnalea J. and Cohen, Bruce N. and Lester, Henry A. (2013) An Engineered Glutamate-gated Chloride (GluCl) Channel for Sensitive, Consistent Neuronal Silencing by Ivermectin. Journal of Biological Chemistry, 288 (29). pp. 21029-21042. ISSN 0021-9258. PMCID PMC3774370. doi:10.1074/jbc.M112.423921. https://resolver.caltech.edu/CaltechAUTHORS:20130909-090111946

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Abstract

A modified invertebrate glutamate-gated Cl− channel (GluCl αβ) was previously employed to allow pharmacologically induced silencing of electrical activity in CNS neurons upon exposure to the anthelmintic drug ivermectin (IVM). Usefulness of the previous receptor was limited by 1) the high concentration of IVM necessary to elicit a consistent silencing phenotype, raising concern about potential side effects, and 2) the variable extent of neuronal spike suppression, due to variations in the co-expression levels of the fluorescent protein-tagged α and β subunits. To address these issues, mutant receptors generated via rational protein engineering strategies were examined for improvement. Introduction of a gain-of-function mutation (L9′F) in the second transmembrane domain of the α subunit appears to facilitate β subunit incorporation and substantially increase heteromeric GluCl αβ sensitivity to IVM. Removal of an arginine-based endoplasmic reticulum retention motif (RSR mutated to AAA) from the intracellular loop of the β subunit further promotes heteromeric expression at the plasma membrane possibly by preventing endoplasmic reticulum-associated degradation of the β subunit rather than simply reducing endoplasmic reticulum retention. A monomeric XFP (mXFP) mutation that prevents fluorescent protein dimerization complements the mutant channel effects. Expression of the newly engineered GluCl opt α-mXFP L9′F + opt β-mXFP Y182F RSR_AAA receptor in dissociated neuronal cultures markedly increases conductance and reduces variability in spike suppression at 1 nm IVM. This receptor, named “GluClv2.0,” is an improved tool for IVM-induced silencing.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1074/jbc.M112.423921 DOIArticle
http://www.jbc.org/content/288/29/21029PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774370/PubMed CentralArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, September 28, 2012, and in revised form, May 27, 2013 Published, JBC Papers in Press, May 29, 2013. This work was supported, in whole or in part, by National Institutes of Health Grants NS034407, EY018502, and MH088550. This work was also supported by the McKnight Foundation. We thank Sheri McKinney for providing hippocampal neuron cultures. We also thank D. A. Dougherty and D. Anderson for comments.
Funders:
Funding AgencyGrant Number
NIHNS034407
NIHEY018502
NIHMH088550
McKnight FoundationUNSPECIFIED
Issue or Number:29
PubMed Central ID:PMC3774370
DOI:10.1074/jbc.M112.423921
Record Number:CaltechAUTHORS:20130909-090111946
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130909-090111946
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:41160
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Sep 2013 20:19
Last Modified:10 Nov 2021 04:26

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