Color-tuned channelrhodopsins for multiwavelength optogenetics

Abstract

Channelrhodopsin-2 is a light-gated ion channel and a major tool of optogenetics. It is used to control neuronal activity via blue light. Here we describe the construction of color-tuned high efficiency channelrhodopsins (ChRs), based on chimeras of Chlamydomonas channelrhodopsin-1 and Volvox channelrhodopsin-1. These variants show superb expression and plasma membrane integration, resulting in 3-fold larger photocurrents in HEK cells compared with channelrhodopsin-2. Further molecular engineering gave rise to chimeric variants with absorption maxima ranging from 526 to 545 nm, dovetailing well with maxima of channelrhodopsin-2 derivatives ranging from 461 to 492 nm. Additional kinetic fine-tuning led to derivatives in which the lifetimes of the open state range from 19 ms to 5 s. Finally, combining green- with blue-absorbing variants allowed independent activation of two distinct neural cell populations at 560 and 405 nm. This novel panel of channelrhodopsin variants may serve as an important toolkit element for dual-color cell stimulation in neural circuits.

FIGURE 1.

Engineering of ChR chimeras with different spectral properties. A, color-coded ChR chimeras. Peak photocurrent amplitudes are qualitatively indicated by +, and peak absorption wavelengths are given numerically. In the cases where two wavelength numbers are listed they reflect two distinct maxima recorded at pH_o 5 and 9, that overlap at pH_o 7 and broaden the spectrum. B, averaged peak photocurrent amplitudes for all high efficiency color-tuned ChRs and their parental origins in HEK cells. C, action spectra of color-tuned ChR variants compared with action spectra of V1 (dark gray) exhibiting a blue shoulder and C1V1 (light gray) (n = 5, 3, 5, 8, 3, and 3 for C2-HR, C2-ET-TC, V1, C1V1-A, C1V1-SG, and C1V1-ET). Amplitudes were linearly corrected to light intensities. D, confocal images of HEK cells expressing color variants and their parental ChRs. E and F, normalized photocurrents upon pulse (in HEK cells) or laser flash stimulation (in Xenopus oocytes) for all color mutants. Photocurrents decay biexponentially after activation for 300 ms whereas off-kinetics of dark-adapted ChRs after laser activation can be fitted by a single exponential. Respective τ_off values and their relative amplitudes are given numerically. G, recovery kinetics of the transient peak in a two-pulse experiment at pH_o 7.2 for color mutants. Between pulses membrane voltage was kept at −60 mV (n = 4, 4, 4, and 3 for C1V1-ET, C2-ET-TC, C2-HR, and C1V1-SG). A typical photocurrent trace is shown as an inset (light blue).

FIGURE 2.

Generation of fast- and slow-cycling color-tuned ChRs. A, structural model of C1V1 based on the three-dimensional structure of C1C2–52 (Protein Data Bank code 3UG9) (23) depicting all relevant amino acid positions. B and C, average peak photocurrent amplitudes of ChRs in HEK cells at their respective peak wavelength for slow- and fast-cycling mutants (>10 mW·mm⁻²). D, normalized current responses to light trains of 10 Hz for fast-cycling mutants. E, stimulus-response curves of peak currents I_P upon 7-ns laser flashes (n = 5, 3, and 4 for C2-TC, C2-CT and C1V1). The inset shows the respective current traces for C2-TC and C2-CT. F, light titration curve for I_S of slow- and fast-cycling mutants upon stimulation with 5-s light pulses (n = 5, 3, and 5 for C2-ET-TC, C2-CS, and C1V1). Inset exemplifies individual traces for C2-CS and C2-ET-TC. G–I, action potential firing in hippocampal neurons. G, at 560-nm light C1V1-ET-ET triggers spikes reliably compared with C2-LC-TC. H, at 405-nm light C2-LC-TC is applicative to evoke spikes at 0.875 mW·mm⁻² whereas no spikes are seen for C1V1-ET-ET at similar intensities. By contrast, at 8.2 mW·mm⁻² C1V1-ET-ET evokes spikes with same probability as C2-LCTC. I, in responding to trains of light pulses up to 20 Hz, both C2-LC-TC and C1V1-ET-ET are spiking with high fidelity. However at 40–50 Hz and above, the probability of successful spike generation is significantly reduced compared with 5 Hz (n > 4) particularly for C1V1-ET-ET.

FIGURE 3.

Dual light excitation and ion selectivity for blue and green light-absorbing ChRs. A, action spectra for C2-TC (blue), C1V1 (gray), and C1V1-triple (orange) (n = 2, 8, and 9). Excitation at 405, 470, and 560 nm is indicated by colored bars. B, responses of C2-TC, C1V1, and C1V1-triple upon 10-ms light pulses at 405 and 560 nm. C, Ca²⁺ response in two mixed cell populations. Membrane potential was adjusted through mTrek potassium channel with extracellular K⁺. Ca²⁺ influx through Ca_V3.2 was monitored by a change in fura-2 fluorescence. HEK cells were separately transfected with C1V1-triple-eCFP and C2-TC-mCherry are shown as fluorescence and transmission overlay (left) and as fura-2 emission (right). Corresponding fura-2 traces for cyan and red fluorescent cells are shown underneath (two trials with 8 and 13 cells for C2-TC and C1V1-triple). D and E, peak photocurrent amplitudes and profiles for C2, C1V1-A, C1V1-B (both exhibit virtually identical profiles), C2-LC, C2-TC, and C2-LC-TC for 300-ms light pulses. F, average initial currents (upper panel) and stationary photocurrents with S.D. (lower panel) at different ionic conditions. Intracellular buffer was kept at 110 mm N-methyl-d-glucamine-Tris, pH_i 9.0. Currents were normalized to I₀ at standard conditions. All data points are evaluated at −60 mV (each n > 8). G, fura-2 fluorescence in ChR-expressing HEK cells after light stimulation for 10 s (black bar) at an extracellular CaCl₂ concentration of 70 mm at pH_o 7.2 (n > 10 cells). Fluorescence intensity before light application is normalized to 1.

FIGURE 4.

Current-voltage relationships for C2, C1V1-A, and C2-LC-TC. Photocurrents were recorded at high Ca²⁺ (70 mm) and variable internal pH (pH_i = 9 for A–C, pH_i = 7.2 for D and E). Reversal potentials of initial currents I₀ (filled circles) and stationary currents I_S (open circles) are indicated by green arrows (n > 8).