Optimization of the GluCl/IVM Neuronal Silencing Tool via Protein Engineering

A variety of genetically encoded tools have been developed for deciphering the neural circuitry of the brain. Such tools allow physical manipulation of neuronal excitability in a reversible, cell-specific manner, enabling researchers to establish how electrical activity and connectivity facilitate the information processing that mediates perception and drives behavior. An expanding toolkit of engineered neuroreceptors, particularly those actuated by orthogonal pharmacological ligands, provide noninvasive manipulation of regional or disperse neuronal populations with adequate spatiotemporal precision and great potential for multiplexing. We previously engineered an invertebrate glutamate-gated chloride channel (GluCl alphabeta) that enabled pharmacologically induced silencing of electrical activity in targeted CNS neurons in vivo by the anthelmintic drug compound ivermectin (IVM; Lerchner et al., 2007). With this receptor, GluCl opt alpha-CFP + opt beta-YFP Y182F, the concentration of IVM necessary to elicit a consistent silencing phenotype was higher than expected, raising concern about its potential side effects. Considerable variability in the extent of spike suppression was also apparent and was attributed to variable co-expression levels of alpha and beta subunits. Thus, a rational protein engineering strategy was employed to optimize the GluCl/IVM tool. To increase agonist sensitivity, a gain-of-function gating mutation involving the highly conserved leucine 9' residue of the alpha pore-lining M2 transmembrane domain was introduced. Various mutations at this position facilitate channel opening in the absence and presence of ligand. Analysis of side chain properties revealed that helix-destabilizing energy correlated with increases in agonist sensitivity. One mutation, L9'F, enhances beta subunit incorporation to substantially increase IVM sensitivity without permitting unliganded channel opening. Removal of an arginine-based ER retention motif (RSR_AAA) from the intracellular loop of beta promoted plasma membrane expression of heteromeric GluCl alphabeta by preventing ER-associated degradation of the beta subunit. An additional monomeric XFP mutation complements these effects. The newly engineered GluCl opt alpha-mXFP L9'F + opt beta-mXFP Y182F RSR_AAA receptor significantly increases conductance and reduces variability in evoked spike generation in vitro using a lower concentration of IVM. This receptor, dubbed 'GluClv2.0', is an improved tool for IVM-induced silencing.