Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

Abstract

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

Keywords: channelrhodopsin; chloride; neuronal inhibition; optogenetics; structure.

Fig. 1.

Design and characterization of iC++ in cultured neurons of rat hippocampus. (A) V_rev of iC1C2 mutations compared with chloride-conducting channelrhodopsins at an intracellular chloride concentration of 4 mM. EN/Q/C: E83N/Q/C; ES: E273S; RH: R134H; SQ: S90Q; 2KR: K117R/K242R. (B) Photocurrent amplitudes measured at the V_AP (−51 ± 4 mV). (C) R_in of channelrhodopsin-expressing cells. (D) Stationary photocurrents per fluorescence of eYFP-tagged constructs expressed in cultured neurons. (E) C1C2 structure depicting iC++ mutations. Numbering refers to N-terminal–truncated channel iC++. Corresponding positions in the original C1C2 structure are indicated in parentheses. (F) Structural model of the putative cytosolic ion gate in C1C2. Histidine 134 (173) (gray) was replaced by arginine in iC1C2 (orange) but not in iC++. (G) Voltage trace of a cultured neuron expressing iC++. APs were evoked by pulsed electrical inputs at 10 Hz (dotted line) and were inhibited by continuous light application (blue bar) for 4 s. (H) Inhibition probability of APs evoked by pulsed current injections (6 s, 10 Hz) with pulse widths of 30 ms (Left), and 5 ms (Right) under 4-s light application. Current inputs were individually titrated to the V_AP. Shorter pulses required stronger current amplitudes. Average: 30 ms: 202 ± 11 pA; 5 ms: 559 ± 40 pA. pH_ext = 7.3, pH_int = 7.2; [Cl⁻]_ext = 147 mM, [Cl⁻]_int = 4 mM for entire figure. ***P < 0.005, ****P < 0.0001. Error bars indicate SEM; all values and numbers are listed in SI Appendix, Table S1.

Fig. 2.

Model for ion selectivity in channelrhodopsins. (A) Structure of the nonselective cation channel C1C2 (Protein Data Bank ID code: 3UG9) (7) with calculated electrostatic potentials of transmembrane helices 1 (TM1) and 7 (TM7), which form the ion-conducting pore together with TM2 and TM3. The electrostatic potential is predominantly negatively charged because of the large number of glutamates facing the pore interior (red: −1 kT/e). Charged groups were considered fully protonated for a simplified first approximation. (B) Structures of C1C2 with replacements for iC++ (Left) and iC1C2 (Right) mutations. The predominantly positive charge of the electrostatic potential of the pore presumably attracts anions and excludes cations (blue: +1 kT/e). (C) C1C2 structure with four replacements that are equivalently found in GtACR2: C1C2-E122S (GtACR2-S57), C1C2-E136T (GtACR2-T67), C1C2-E140A (GtACR2-A71), and C1C2-E162S (GtACR2-S93). These replacements presumably contribute to the formation of a positively charged, anion-conducting pore.

Fig. 3.

Biophysical characterization of iC++ in HEK293 cells. (A) iC++ photocurrents recorded at membrane potentials from −80 to +40 mV during light application (blue bar). Peak currents (I₀) decay to smaller stationary level (I_S). (B and C) Amplitudes (B) and V_rev (C) of iC++ peak and stationary currents with 150 mM external ([Cl⁻]_ext) and varying internal [Cl⁻]_int chloride concentrations. The Nernst equilibrium for chloride (E_Cl) is indicated for each condition (green bar). (D) Change in R_in in iC1C2- and iC++-expressing HEK cells in the dark when [Cl⁻]_ext was increased from 10 to 150 mM and [Cl⁻]_int was fixed at 10 mM. Values are normalized to [Cl⁻]_ext = 10 mM (black line). iC++ does not conduct ions in the dark. (E) pH-dependent photocurrents of iC1C2 and iC++ at neutral (black, pH_ext 7.2) and acidic (red, pH_ext 5.0) external pH values, pH_int 7.2. Amplitudes at different pH values were recorded from the same cells. (F) Photocurrent at pH_ext 5.0/0 mV normalized to values at pH_ext 7.2/0 mV (black line). (G) V_revs from E. ****P < 0.0001. Error bars indicate SEM; all values and numbers are listed in SI Appendix, Table S2.

Fig. 4.

Chloride-dependent inhibition of pyramidal neurons in mouse mPFC. (A) Confocal image of iC++ expression in mPFC 5 wk after virus injection. (Scale bar: 100 µm.) (B and C) V_revs (B) and photocurrents (C) of iC++ at the V_AP (−52 ± 1 mV) measured for 5 s with light application under varying internal chloride concentrations ([Cl⁻]_int: 4, 12, and 20 mM). Average V_rest = −80 mV ± 2. (D) Voltage trace of an iC++-expressing neuron showing AP generation by continuous current injections (black bar) and inhibition during 10-s light application (blue bar). (E) Spike frequency before (0.5 s), during (10 s), and after (1 s) light application under varying internal chloride concentrations. Continuous current injections were titrated individually to reach the V_AP; average: 229 ± 24 pA. n.s.: P > 0.05; ****P < 0.0001. Error bars indicate SEM; all values and numbers are listed in SI Appendix, Table S3.

Fig. 5.

SwiChR++ characterization in HEK293 cells and neurons from acute mPFC slices. (A) Photocurrent traces of iC++ (red) and SwiChR++ (black) showing the time course of the channel closure. (Left) Traces were recorded in HEK293 cells at 0 mV in response to brief application of blue light (blue arrow). (Right) The same recordings at higher time resolution, depicting the biexponential channel closure of iC++ with a dominant fast component (τ_fast) and a slow component (τ_slow). (B) Photocurrent of SwiChR++ upon the application of blue light (blue bar) and delayed red light (red bar), which accelerates channel deactivation. (C, Left) Tau values for biexponential channel closure of iC++. (Right) Amplitude ratios for the fast (τ_fast) and slow (τ_slow) components. (D) Tau values for the monoexponential channel closure of SwiChR++ in the dark and with the application of light (600 nm). (E) Confocal image of SwiChR++ expression in mouse mPFC 5 wk after virus injection. (Scale bar: 100 µm.) (F) Voltage trace of an SwiChR++-expressing pyramidal neuron showing APs evoked by continuous current injections (black bar) and inhibition for 10 s upon light application for 1 s (blue bar). Spiking recovers upon red light stimulation (red bar). (G) Spike frequency before (0.5 s), during (10 s), and after (2 s) SwiChR activation at varying intracellular chloride concentrations. Input currents were titrated individually to reach the V_AP; average: 205 ± 27 pA. n.s.: P > 0.05; ****P < 0.0001. Error bars indicate SEM; all values and numbers are listed in SI Appendix, Table S4.

Fig. 6.

Place aversion induced by inhibition of dopaminergic VTA neurons in mice. (A) An AAV encoding a Cre-dependent opsin (iC++, eNpHR3.0, or eArch3.0 n = 4 per group) was injected into the VTA of DAT:Cre mice, and optical fibers were implanted dorsal to this area. (B, Upper) Schematic of the real-time place-aversion task. Mice were allowed to explore a two-compartment chamber freely. Entry into one compartment triggered continuous illumination (473 nm for iC++; 532 nm for eNpHR3.0 and eArch3.0) as long as the mouse remained in the light-paired compartment. The test was repeated, with distinct contextual cues to minimize generalization, at multiple light intensities. (Lower) Representative tracking data from the 5-mW experiment demonstrating robust avoidance of the light-paired compartment. (C) Percent time spent in the light-paired compartment at various light intensities. No side preference was observed when the light intensity was 0 mW (light-off control), but at 0.5 mW and 5 mW all groups avoided the light-paired compartment.***P < 0.005. Error bars indicate SEM; all values and numbers are listed in SI Appendix, Table S5.

Fig. 7.

Using inhibitory channelrhodopsins to silence neurons involved in the engram supporting fear memory. (A) Robust, localized transgene expression (green) following microinjection of HSV-CREB/iC++-YFP vector into the LA. (Scale bar: 100 μm.). (B) Cartoon of experimental methods used to test the behavioral effects of inhibitory opsins. Mice were microinjected with viral vectors expressing CREB/iC++, CREB/iC1C2, or CREB/eNpHR3.0 into the LA before auditory fear conditioning. Neurons overexpressing CREB are preferentially allocated (red) to the engram supporting the resulting auditory fear memory. Using light to silence these neurons optogenetically results in decreased freezing, indicating impaired memory retrieval. (C) Neurons overexpressing CREB before training are preferentially allocated to the engram that supported fear memory. Silencing these neurons using iC++, iC1C2, or eNpHR3.0 reversibly disrupted memory retrieval. Disrupting the activity of the same number of neurons that are not allocated to the fear engram (those not overexpressing CREB) did not impair memory retrieval. n.s.: P > 0.05; ***P < 0.005. Error bars indicate SEM; all values and numbers are listed in SI Appendix, Table S6.