Enhancement of the long-wavelength sensitivity of optogenetic microbial rhodopsins by 3,4-dehydroretinal - PubMed (original) (raw)

. 2012 Jun 5;51(22):4499-506.

doi: 10.1021/bi2018859. Epub 2012 May 22.

Affiliations

Enhancement of the long-wavelength sensitivity of optogenetic microbial rhodopsins by 3,4-dehydroretinal

Oleg A Sineshchekov et al. Biochemistry. 2012.

Abstract

Electrogenic microbial rhodopsins (ion pumps and channelrhodopsins) are widely used to control the activity of neurons and other cells by light (optogenetics). Long-wavelength absorption by optogenetic tools is desirable for increasing the penetration depth of the stimulus light by minimizing tissue scattering and absorption by hemoglobin. A2 retinal (3,4-dehydroretinal) is a natural retinoid that serves as the chromophore in red-shifted visual pigments of several lower aquatic animals. Here we show that A2 retinal reconstitutes a fully functional archaerhodopsin-3 (AR-3) proton pump and four channelrhodopsin variants (CrChR1, CrChR2, CaChR1, and MvChR1). Substitution of A1 with A2 retinal significantly shifted the spectral sensitivity of all tested rhodopsins to longer wavelengths without altering other aspects of their function. The spectral shift upon substitution of A1 with A2 in AR-3 was close to that measured in other archaeal rhodopsins. Notably, the shifts in channelrhodopsins were larger than those measured in archaeal rhodopsins and close to those in animal visual pigments with similar absorption maxima of their A1-bound forms. Our results show that chromophore substitution provides a complementary strategy for improving the efficiency of optogenetic tools.

PubMed Disclaimer

Figures

FIGURE 1

FIGURE 1

Absorption spectra of E. coli cells expressing the proton pump AR-3 upon reconstitution with A1 or A2 retinal.

FIGURE 2

FIGURE 2

Photoinduced electrical signals by AR-3 expressed in E. coli in the presence of A1 (black traces) or A2 (red traces) retinal. Solid lines, current traces; dashed lines; transmembrane charge transfer (calculated as area under the current traces). Both sets of curves were normalized for easier kinetics comparison.

FIGURE 3

FIGURE 3

Action spectra of charge movement by AR-3 expressed in E. coli cells (solid symbols and lines) or HEK293 cells (open symbols, dashed lines) in the presence of A1 retinal (black symbols and lines) or A2 retinal (red symbols and lines). Errors bars were of the same magnitude for all spectra, but for presentation purposes are shown only for one of them.

FIGURE 4

FIGURE 4

(A) Action spectra of photocurrents generated by _Ca_ChR1 from C. augustae in HEK293 cells incubated with A2 retinal (solid symbols, solid lines), or A1 retinal (open symbols, dashed line, adopted from (20)). Final concentration of A2 retinal was 5 (solid triangles) or 25 (solid squares) μM, that of A1 retinal was 2.5 μM. (B) Absorption spectra of purified _Ca_ChR1 expressed in Pichia cells in the presence of 5 μM of A2 (solid line) or A1 retinal (dashed line).

FIGURE 5

FIGURE 5

Reconstitution spectra of _Ca_ChR1 in bleached Pichia membranes measured after the addition of A1 retinal (black symbols and line), a mixture of A1 and A2 retinal (green symbols and line), or A2 retinal (red symbols and line). Symbols, experimental data points; lines, 15 nm FFT smoothing of the data. For other details see text.

FIGURE 6

FIGURE 6

Action spectra of photoinduced currents generated by _Cr_ChR2 in HEK293 cells after incubation with A2 retinal (solid symbols, solid line), or A1 retinal (open symbols, dashed line).

FIGURE 7

FIGURE 7

(A) Photoelectric currents generated by _Cr_ChR2 in HEK293 cells incubated with A2 retinal in response to high-intensity stimuli (~1022 photons × m2 × s−1) at 520 nm. (B) Comparison of the current kinetics at low intensity (<1020 photons × m2 × s−1) in response to 440 nm light, mostly absorbed by the A1-bound pigment (black line), and 530 nm light, mostly absorbed by the A2-bound pigment (red line).

FIGURE 8

FIGURE 8

Action spectra of photocurrents generated by _Cr_ChR1 (A) or _Mv_ChR1 (B) in HEK293 cells incubated with A2 retinal (solid symbols, solid lines), or A1 retinal (open symbols, dashed lines, adopted from (20) and (19), respectively).

FIGURE 9

FIGURE 9

(A) Extension of the long wavelength spectral boundary of the proton pump AR-3 and various channelrhodopsins by incubation with A2 retinal. Bars show the spectral bands with more than 1/e of maximal efficiency for the pigments formed with A1 (blue) or A2 (red) retinal. The absorption spectrum of hemoglobin (oxidized + reduced) is shown for comparison. (B) Theoretical estimation of the total number of actinic photons absorbed over the visible range by the tested rhodopsins at different depths of brain tissue (for more details see text). Solid symbols and lines, pigments formed with A2 retinal; open symbols and dashed lines, pigments with A1 retinal. Black, AR-3; red, _Cr_ChR1; green, _Cr_ChR2; blue, _Ca_ChR1; cyan, _Mv_ChR1.

References

    1. Spudich JL, Yang C-S, Jung K-H, Spudich EN. Retinylidene proteins: structures and functions from archaea to humans. Annu. Rev. Cell Dev. Biol. 2000;16:365–392. - PubMed
    1. Chow BY, Chuong AS, Klapoetke NC, Boyden ES. Synthetic physiology strategies for adapting tools from nature for genetically targeted control of fast biological processes. Methods Enzymol. 2011;497:425–443. - PMC - PubMed
    1. Deisseroth K. Optogenetics. Nat. Methods. 2011;8:26–29. - PMC - PubMed
    1. LaLumiere RT. A new technique for controlling the brain: optogenetics and its potential for use in research and the clinic. Brain Stimul. 2011;4:1–6. - PubMed
    1. Schoenenberger P, Scharer YP, Oertner TG. Channelrhodopsin as a tool to investigate synaptic transmission and plasticity. Exp. Physiol. 2011;96:34–39. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources