High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels

Abstract

Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise induction of action potentials with short light pulses. A limiting factor for many optophysiological experiments is the relatively small photocurrent induced by ChR2. We screened a large number of ChR2 point mutants and discovered a dramatic increase in photocurrent amplitude after threonine-to-cysteine substitution at position 159. When we tested the T159C mutant in hippocampal pyramidal neurons, action potentials could be induced at very low light intensities, where currently available channelrhodopsins were unable to drive spiking. Biophysical characterization revealed that the kinetics of most ChR2 variants slows down considerably at depolarized membrane potentials. We show that the recently published E123T substitution abolishes this voltage sensitivity and speeds up channel kinetics. When we combined T159C with E123T, the resulting double mutant delivered fast photocurrents with large amplitudes and increased the precision of single action potential induction over a broad range of frequencies, suggesting it may become the standard for light-controlled activation of neurons.

Fig. 1.

Biophysical characterization of photocurrents in Xenopus oocytes. (A) Homology model of the ChR2 retinal binding pocket based on bacteriorhodopsin X-ray data (Protein Data Bank ID Code 1C3W). The chromophore is shown in magenta; residues that have been replaced in this study are labeled in red. (B) Typical photocurrents in oocytes excited by a 1-s light pulse (500 nm, green bar) measured at −100 mV. During light stimulation, the peak current (I_peak) decays to a lower stationary level (I_stationary). The off-kinetics (τ_off) were extracted from tail currents. (C) Stationary photocurrents of single and double mutants (I_stationary), normalized to the reference values of wt ChR2 (I_wt, black; n = 15). HR (green; n = 9), TC (magenta; n = 9), ET/TC (blue; n = 13), EA/TC (red; n = 9), and HR/TC (gray; n = 8). (D) Channel closure (τ_off) of ET/TC (blue) was as fast as wt ChR2 (black); all other tested mutants were significantly slower. (E) Inactivation under continuous light conditions (ratio of I_stationary to I_peak) of TC and ET/TC was similar to wt ChR2. In HR, ET/HR, EA/TC, and HR/TC, inactivation was significantly reduced. ***P < 0.005; *P < 0.05.

Fig. 2.

Voltage-dependence of channel kinetics and spectral properties of ChR2 mutants. (A) Photocurrents after laser flash activation (10 ns; green arrow) measured at −50 mV, normalized to the peak. ET/HR and ET/TC were significantly faster than wt ChR2, whereas HR and TC were significantly slower.***P < 0.005. (B) Photocurrents after laser flash activation at +50 mV. TC, HC, and wt ChR2 slowed down considerably at this membrane potential, whereas ET/HR and ET/TC retained their fast kinetics. (C) To quantify the voltage-dependence of channel kinetics, flash-to-peak and τ_off were analyzed at different membrane potentials (n = 10 cells for each mutant). (D) Time-dependent recovery of peak currents (I_peak) was measured under physiological conditions at −75 mV in oocytes. Recovery was defined as the ratio of ΔI₂ (I_peak2 − I_stationary2) to ΔI₁ (I_peak1 − I_stationary1) and plotted against the interpulse interval. Recovery time constants (τ_rec) of HR (green; n = 3) and ET/TC (blue; n = 3) were significantly faster than wt ChR2 (black; n = 15), whereas TC (magenta; n = 3) was slower. ***P < 0.005; *P < 0.05. (E) IV curves show that the typical inward rectification of wt ChR2 (black) is retained in all mutants. Reversal potentials (V_reversal) were close to zero under physiological conditions (Inset; n = 10, 10, 12, 8). (F) Action spectra measured in hippocampal neurons show red-shifted wavelength optimum of ET/TC (blue curve, n = 13) relative to HR (green curve, n = 10) and TC (red curve, n = 11).

Fig. 3.

Photocurrents of ChR2 variants in hippocampal pyramidal neurons. (A) Neurons in a sparsely transfected rat organotypic slice culture expressing TC and dimeric RFP. (B) Contrast-inverted two-photon images of individual transfected pyramidal neurons. (Scale bar: 50 μm.) (C Left) Photocurrents evoked by 500-ms blue laser illumination (42 mW/mm²). Stationary photocurrents were quantified at the end of the stimulation pulse (dashed line) in neurons that were electrically isolated by blocking excitatory synaptic input with NBQX and dCPP. Escape APs were cropped for clarity (dotted line). (Right) Quantification of stationary photocurrents (n = 9, 11, 14, 13, and 16 for wt, HR, TC, ET/TC, and ET/HR, respectively). Circles depict measurements from individual cells.

Fig. 4.

Stimulation performance of TC variants at 1–100 Hz. (A Left) Whole-cell current-clamp recordings from pyramidal neurons stimulated with 60 brief (2 ms) light pulses at 40 Hz at high laser intensity (42 mW/mm²). Light-evoked activity was isolated by blocking excitatory synaptic input. (Right) Stimulation of the same cells with low (1.9 mW/mm²) light power. (B) Summary of the firing success rates from 1 to 100 Hz at different stimulation intensities (n = 9, 9, 12, and 13 for wt, HR, TC, and ET/TC, respectively). Note the high stimulation efficacy of TC and ET/TC even at low light intensities.

Fig. 5.

Precision of single AP induction. (A Left) With high stimulation light intensity, TC often evoked several spikes in response to a single 2-ms light pulse during 1-Hz stimulation. (Right) Multiplet firing is observed mostly with TC and is reduced at lower stimulation intensity. (B) Multiplet firing is largely restricted to 1-Hz stimulation and rarely occurs at higher stimulation frequencies. (C Left) Example spike trains from neurons expressing TC or ET/TC stimulated with 60 2-ms pulses at 40 Hz with 42 mW/mm² light intensity. Plateau potentials were measured between baseline and the minimal membrane depolarization during the last 500 ms of the stimulation train (dashed lines). (Right) Summary of plateau potentials for 1- to 100-Hz stimulation (n = 9, 11, 14, 13, and 12 for wt, HR, TC, ET/TC, and ET/HR, respectively). (D) Plateau depolarization at 100 Hz was highly correlated with the product of photocurrent amplitude and channel closing speed.

Fig. 6.

Evaluation of different ChR2 variants for use in pyramidal neurons. Photocurrent amplitude (same data as Fig. 3C) and channel speed, defined as (flash to peak + τ_off,)⁻¹ (same data as Fig. 2C) determine the performance in neurons. Bright circles, kinetics at −75 mV; pale circles, at −50 mV. The ideal ChR should gate large currents with rapid kinetics (green shaded corner).