Structure-guided transformation of channelrhodopsin into a light-activated chloride channel

Abstract

Using light to silence electrical activity in targeted cells is a major goal of optogenetics. Available optogenetic proteins that directly move ions to achieve silencing are inefficient, pumping only a single ion per photon across the cell membrane rather than allowing many ions per photon to flow through a channel pore. Building on high-resolution crystal-structure analysis, pore vestibule modeling, and structure-guided protein engineering, we designed and characterized a class of channelrhodopsins (originally cation-conducting) converted into chloride-conducting anion channels. These tools enable fast optical inhibition of action potentials and can be engineered to display step-function kinetics for stable inhibition, outlasting light pulses and for orders-of-magnitude-greater light sensitivity of inhibited cells. The resulting family of proteins defines an approach to more physiological, efficient, and sensitive optogenetic inhibition.

Fig. 1. Rational design and screen: V_rev-shifted ChRs

(A) C1C2 crystal structure [Protein Data Bank (PDB) 3UG9] (24), with residues used for screening in blue (retinal chromophore in magenta). (B) C1C2 mutations screened in neurons for photocurrent size at −80 mV (n = 6 to 8 cells). Arrows indicate nine mutations selected for C1C2_5x (T98S/E129S/E140S/E162S/T285N) and C1C2_4x (V156K/H173R/V281K/N297Q) constructs. (C) V_rev of C1C2 single-mutation constructs (n = 6 to 8 cells). (D) Comparison of photo-current sizes for C1C2, C1C2_4x, and C1C2_5x. (E) Comparison of V_rev for C1C2, C1C2_4x, and C1C2_5x (n = 8 to 10 cells). Error bars indicate SEM.

Fig. 2. iC1C2: biophysical properties

(A) C1C2 structure, with the nine residues mutated in C1C2_4x and C1C2_5x in orange. (B) Representative photocurrents and (C) corresponding current-voltage relationships recorded at membrane potentials from −75 mV to +55 mV upon 475 nm light activation (power density, 5 mW/mm²). (D) V_rev of C1C2, iC1C2, C1C2_4x, and C1C2_5x [neuronal recording solutions are available in (35)]. (E) Activation spectra of NpHR, C1C2, and iC1C2 measured at power density 0.65 mW/mm² for each wavelength and normalized to the maximum amplitude (n = 6 cells). (F) V_rev of C1C2, iC1C2, C1C2_4x, and C1C2_5x, with internal (int) 120 mM KCl and external (ext) 120 mM NaCl, CsCl, or NaGluconate, pH 7.3, characterized in HEK cells. (G) As in (A), with ext 120 mM NaCl and int 120 mM KCl, CsCl, or KGluconate, pH 7.3 (n = 6 to 17 cells). (H) V_rev of iC1C2 under equal (eq) external and internal pH, generating a Nernst potential for protons of 0 mV (dotted green line) at pH 6 and 7.3. [Cl⁻]_i concentration was 8 mM, and [Cl⁻]_o was 128 mM, generating a Nernst potential for Cl⁻ of −71 mV (dotted red line) (n = 6 to 9 cells). (I) Current-voltage relationship recorded with equal external and internal pH values at pH 6 and 7.3; internal and external [Cl⁻] of 8 mM and 128 mM, respectively (n = 3 to 8 cells). (J) Photocurrent of iC1C2 at 0 mV from the current-voltage relationship in (I). Error bars indicate SEM.

Fig. 3. Characterization of iC1C2 in mammalian neurons

(A) Representative photocurrents of C1C2 (left) and iC1C2 (right) recorded at membrane potentials ranging from −80 to 0 mV. 475 nm light (blue bar) was applied at 5 mW/mm². (B) Corresponding current-voltage relationship for photocurrents. (C) V_rev of C1C2 and iC1C2 relative to threshold for spike generation (V_AP) and to neuron resting potential (V_rest) (n = 8 to 22 cells). (D) Photocurrent amplitudes (left) and membrane polarization at V_AP (right) (n = 9 to 14 cells). (E) Mean changes in input resistances were normalized to pre-light value (n = 9 to 20 cells). (F) Sample voltage traces of iC1C2-expressing neuron stimulated with varying current injections for 1.5 s, and additional 0.5 s, 475 nm, 5 mW/mm² pulses showing effective clamping toward V_rev: shown are hyperpolarizing responses below V_AP. Error bars indicate SEM.

Fig. 4. Fast and bistable inhibition of neuronal spiking with iC1C2 and SwiChR

(A and B) Representative voltage traces of iC1C2-expressing neurons stimulated with either (A) a continuous electrical pulse (3s) or (B) pulsed current injections (10Hz/3s). Electrically evoked spikes were inhibited by 475 nm of light (blue bar) at 5 mW/mm². (C) Distribution of spike-inhibition probability for iC1C2-expressing cells (n = 18 neurons; fraction of spikes blocked shown). (D) Inward and outward photocurrents of SwiChR_CT in HEK cell upon 475 nm of light (blue bar). (E) Channel off-kinetics (τ) for iC1C2, SwiChR_CT, and SwiChR_CT exposed to red light during channel closure. (F) Light sensitivity of SwiChR_CT compared with that of iC1C2 and NpHR. iC1C2 and SwiChR_CT were activated with 470 nm, and NpHR was activated with 560 nm. Photocurrents were measured at light intensities between 0.0036 and 5 mW/mm², and holding potential was −80 mV. Amplitudes were normalized to the maximum value for each construct (n = 6 to 8 cells). (G) Reversal potential of iC1C2, SwiChR_CT, and SwiChR_CA relative to V_AP and V_rest (n = 10 to 22) (left). Photocurrent amplitudes at V_AP are shown at right (n = 9 to 15 cells). (H) Bistable spiking modulation by SwiChR_CT. Spiking was induced with a continuous electrical pulse (3 s) and stably inhibited with 475 nm light (blue bar). Spiking resumed after 632 nm of light application (red bar). Light power density was 5 mW/mm². Error bars indicate SEM.