title: An engineered glutamate-gated chloride (GluCl) channel for sensitive, consistent neuronal silencing by ivermectin.
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.
authors.affiliation: From the Biochemistry and Molecular Biophysics Option and; the Division of Biology, California Institute of Technology, Pasadena, California 91125.
authors.forename: Shawnalea J
authors.initials: SJ
authors.lastname: Frazier
authors.affiliation: the Division of Biology, California Institute of Technology, Pasadena, California 91125.
authors.forename: Bruce N
authors.initials: BN
authors.lastname: Cohen
authors.affiliation: the Division of Biology, California Institute of Technology, Pasadena, California 91125. Electronic address: lester@caltech.edu.
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D007559:Ivermectin / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D015202:Protein Engineering; D055503:Protein Multimerization / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D029742:Caenorhabditis elegans Proteins; D018118:Chloride Channels; D008164:Luminescent Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; C109777:glutamate-gated chloride channels; D018698:Glutamic Acid; D007559:Ivermectin
keywords: Arginine-based ER Retention Motif; Cys-loop Receptors; Electrophysiology; GluCl Channel; Ion Channels; Ivermectin; Neuroscience; Neurotransmitter Receptors; Silencing