NEUROSCIENCE. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics

Abstract

Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

Fig. 1. Phylogeny and photoactivity of G. theta ACRs

(A) Phylogenetic tree of CCRs and ACRs. (B and C) ClustalW alignments of transmembrane helices 2 (B) and 3 (C) Abbreviated organism names are: Gt Guillardia theta; Cr Chlamydomonas reinhardtii; Ca Chlamydomonas augustae; Mv Mesostigma viride; Hs, Halobacterium salinarum; Nm, Nonlabens marinus. The last residue numbers are shown on the right. Conserved Glu residues in helix 2 are highlighted in yellow, Glu residues in the position of bacteriorhodopsin Asp85 in red, and His residues corresponding to His134 of CrChR2 in blue. (D) Photocurrents of GtACR1, GtACR2, and CrChR2 in HEK293 cells in response to a saturating light pulse at −60 mV. (Inset) Mean amplitudes of peak (solid bars) and stationary (hatched bars) currents (n = 18 to 20 cells). (E) Dependence of the peak and stationary current amplitudes and rise rates on stimulus intensity. (F) Action spectra of photocurrents.

Fig. 2. ACRs do not conduct cations

Photocurrents generated by GtACR1 (A) and GtACR2 (B) in HEK293 cells at the membrane potentials changed in 20-mV steps from −60 mV at the amplifier output (bottom to top). The pipette solution was standard, and the bath solution was as indicated. (C) IE relationships measured at various pH of the bath. The data (mean values ± SEM, n = 4 to 6 cells) were corrected for liquid junction potentials (table S1) and normalized to the value measured at −60 mV at pH 7.4. Representative data for CrChR2 are shown for comparison. (D) E_rev shifts measured upon variation of the cation composition of the bath. The data are mean values ± SEM (n = 3 to 6 cells).

Fig. 3. Anion selectivity of ACRs

Photocurrents generated by GtACR1 (A) and GtACR2 (B) in HEK293 cells at the membrane potentials changed in 20-mV steps from −60 mVat the amplifier output (bottom to top). The pipette solution was standard, and the bath solution was as indicated. (C) IE relationships measured at various Cl⁻ concentrations in the bath. The data (mean values ± SEM, n = 4 to 6 cells) were corrected for liquid junction potentials (table S1) and normalized to the value measured at −60 mV at 156 mM CI⁻. The dashed vertical lines show the Nernst equilibrium potential for Cl⁻ at the bath concentrations used. (D) E_rev shifts measured upon variation of the anion composition of the bath. The data are mean values ± SEM (n = 3 to 6 cells).

Fig. 4. GtACR2 as a hyperpolarizing tool

(A) Light-intensity dependence of photocurrents generated by GtACR2, slow ChloC, and Arch in HEK293 cells at 20 mV. The arrows show the difference in light sensitivity. (B) IE relationship for GtACR2 in neurons. The data (mean values ± SEM, n = 5 cells) were corrected for LJP (table S2). The dashed vertical line shows the resting potential (E_rest). The ranges of activity for Cl⁻-conducting ChR mutants are from (14, 15). (C) Photoinhibition of spiking induced by pulsed current injection in a typical neuron expressing GtACR2. The light intensity was 0.026 mW/mm². (D) The dependence of the rheobase of current ramp-evoked spikes on the light intensity in a typical neuron expressing GtACR2. The data are mean values ± SEM (n = 5 repetitions). Light was applied 0.1 s before the beginning of the current ramp. (Inset) Comparative efficiency of GtACR2 and the ChloC mutants represented as a reciprocal of the minimal light intensity sufficient to fully suppress spiking. The data for GtACR2 are the mean value ± SEM (n = 7 neurons). Data for the ChloC mutants under continuous illumination are from (14). (E) Kinetics of the photocurrents generated by GtACR2 in response to a 1-s light pulse and by slow ChloC in response to a 15-s light pulse (light intensity for both traces was 0.002 mW/mm²). The time constants (τ) were determined by single exponential fits of the recorded traces. The fitted curves are shown as thick lines of the same color as the data. (F) The light-intensity dependence of slow ChloC current amplitude measured at different times after the start of illumination. Data are mean values ± SEM (n = 5 cells). The arrow shows the increase in the light intensity necessary to reach the same current amplitude at 0.1 as at 15 s illumination.