Mutation of the Highly Conserved Pore-Lining Leucine Residue Increases Agonist Sensitivity of a Glutamate-Gated Chloride Channel

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The glutamate-gated chloride channel (GluCl) is an invertebrate, ligand-gated anion channel of the Cys-loop receptor family. It is activated by the endogenous neurotransmitter L-glutamate and by the antiparasitic drug ivermectin. The crystal structure of the Caenorhabditis elegans GluCl alpha homopentamer (3.3A resolution; Hibbs & Gouaux, 2011) shows the location of the glutamate binding site, the separate ivermectin site, and the highly conserved leucine residue at the 9’ position of the pore-lining M2 transmembrane domain. Mutation of this L9’ residue in other Cys-loop receptors dramatically increases agonist sensitivity. Using whole-cell patch clamp, we found that six of seven mutations at this position (L9's, A, F, I, T, V, but not G) increased the glutamate sensitivity of the heteromeric GluCl channel by factors of 5- to 80-fold. Beta-branched amino acids (Ile, Thr, Val) gave the greatest reductions in EC50. Analysis of side chain properties revealed that helix-destabilizing energy correlated with increased glutamate sensitivity. Except for WT and the L9’F mutation, we were unable to detect glutamate- and/or ivermectin-induced changes in membrane potential using a fluorescent membrane potential assay (FlexStation, Molecular Devices), probably because the spontaneous activity of the mutant channels obscured the fluorescence changes. However, whole-cell patch clamp and fluorescent membrane potential experiments confirmed that the L9’F mutation increased both the glutamate and ivermectin sensitivity of the GluCl channel. Increasing GluCl sensitivity to ivermectin will benefit its use as a neuronal silencing tool.