Recent advances in engineering microbial rhodopsins for optogenetics

Abstract

Protein engineering of microbial rhodopsins has been successful in generating variants with improved properties for applications in optogenetics. Members of this membrane protein family can act as both actuators and sensors of neuronal activity. Chimeragenesis, structure-guided mutagenesis, and directed evolution have proven effective strategies for tuning absorption wavelength, altering ion specificity and increasing fluorescence. These approaches facilitate the development of useful optogenetic tools and, in some cases, have yielded insights into rhodopsin structure-function relationships.

Figure 1. Rhodopsins can be used as actuators and sensors in optogenetics

Actuators transport ions across the membrane to activate or repress neuronal activity. ChRs transport positively charged ions into the cell, while proton-pumping rhodopsins (PPRs) move protons out of the cell. In the ideal case, engineered rhodopsin sensors emit light as fluorescence in the farred in a voltage-dependent fashion.

Figure 2. Residues that affect ion selectivity in the channelrhodopsin C1C2

The illustration shows crystal structure of C1C2, with putative ion gating residues S102, E129 and N297 highlighted in green. Mutation of the gating residue N297 to D results in a significant increase in selectivity for Ca²⁺, while mutation of E129 to Q or A results in a significant decrease in the channel's Ca²⁺ selectivity [20]. Mutating the highly conserved gating residue E129 [45] has significant effects on the channel's selectivity for Cl⁻ in both the C1C2 backbone and the ChR2 backbone (position E90 in the ChR2 backbone) [36,37]. Mutation of E90 in ChR2 to R or K increases the reversal potential as a result of increased Cl⁻ selectivity to generate a light activated inhibitory channel [37]. Residues outside of the putative ion gate also influence channel selectivity (residues highlighted in purple). Mutations Q95A, E162 and D292A have all been shown to enhance H⁺ selectivity. Mutants K132A and Q95A display increased K⁺ permeability in the C1C2 backbone [20].

Figure 3. Bifunctional constructs for all-optical electrophysiology

Archer, an engineered Archaerhodopsin-3 variant, enables optical monitoring of voltage with red light, and perturbation of membrane potential with blue light (left) [8]. Alternatively, one rhodopsin can be used for sensing with red light, while an engineered ChR can be used for perturbing the membrane with blue light (right) [9].