The form and function of channelrhodopsin

Abstract

Channelrhodopsins are light-gated ion channels that, via regulation of flagellar function, enable single-celled motile algae to seek ambient light conditions suitable for photosynthesis and survival. These plant behavioral responses were initially investigated more than 150 years ago. Recently, major principles of function for light-gated ion channels have been elucidated by creating channelrhodopsins with kinetics that are accelerated or slowed over orders of magnitude, by discovering and designing channelrhodopsins with altered spectral properties, by solving the high-resolution channelrhodopsin crystal structure, and by structural model-guided redesign of channelrhodopsins for altered ion selectivity. Each of these discoveries not only revealed basic principles governing the operation of light-gated ion channels, but also enabled the creation of new proteins for illuminating, via optogenetics, the fundamentals of brain function.

Fig. 1. The light-gated pore

The crystal structure of C1C2 is shown as a 3UG9¹³-derived pore snapshot calculated via molecular dynamics (MD). Protonated retinal Schiff base (RSBH+, orange) and key pore (brown) or RBP (green) residues showing protonation states of polar residues (81) are shown in stick form (red, oxygen; blue, nitrogen; white, polar protons). ChR2 residue numbering is used. Displayed waters invaded from both membranes during calculations (81). Two Na⁺ ions (orange circles) are shown at positions of preferred occupation within the access channel and near inner gate calculated by MD (81). Blue arrows denote the presumptive permeation pathway. The two-headed orange arrow shows the predicted (43) tilting dynamic of helix 2. Conserved glutamates in helix 2 are designated E1 to E5.

Fig. 2. Deep roots in botany

(A) Faminzin, pioneer of algal behavior (4). (B) Faminzin’s landmark observation (5): At low light intensities (bottom), Chlamydomonas exhibits phototaxis toward light, whereas at higher light intensities (top) the algal cells accumulate at optimal irradiance. (C) Adapted cartoons showing subcellular Chlamydomonas structure: flagellar beating pattern (20) and eye structure reconstructed from high-resolution tomography (17). Eyespot overlaying part of plasma membrane (arrow) corresponds to ChR location. Layers of carotenoid-containing vesicles (colored spheres) held by chloroplast membranes (green) serve as an optical device (interference reflector) (4, 19).

Fig. 3. Chromophore states and channel currents

Chromophore configurations in RSB: all-trans, 15-anti (trans, anti) of dark-adapted state; 13-cis, 15-syn (cis, syn) of second dark state (increasingly occupied after blue illumination during photocurrent inactivation from initial I₀ to stationary I_S; inset) (1, 37). In corresponding open states occupied after ¹³C=¹⁴C photoisomerization, RSB is in the 13-cis, 15-anti (cis, syn) configuration in the open conducting state O1 and 13-trans, 15-syn (trans, syn) in O2 (1, 37). Cis-trans isomerization is indicated by red arrows, anti-syn isomerization by blue arrows, photochemical conversions by green arrows, and thermal conversions by black arrows.

Fig. 4. Diverse modes of photon-spike transduction logic with underlying structural design

(A) Snapshot of ChR active site (MD calculation from C1C2 structure 3UG9, with predicted repositioning of side chains resulting from Glu¹²³ → Thr (E123T) mutation in red lettering; E123Tcauses inward flipping of Glu⁹⁰ to compensate for the lost Glu¹²³ counterion (43) of the RSBH⁺, additionally preventing inactivation. (B) Photon-spike transduction mode arising from ChETA mutation [ChR2-E123Tvariant (45)]: single blue flash–single spike coupling with high speed and high fidelity. Pore redesign implements faster closure after light-off, permitting rapid firing [e.g., 200-Hz trains in interneurons (45)]. (C) Pore residues (from C1C2 structure 3UG9) altered in spectral and selectivity variants. Modification of inner gate in red-activated C1V1-E83Tvariant (magenta letter–designated mutation; ChR2 numbering) (54). Selectivity variants are shown as original cation-conducting C1C2 pore residues and modifications to create the Cl⁻-selective iC++ [new pink side chains overlaid on original C1C2 green side chain positioning; blue letters denote iC++ mutations (62, 64)] or iChloC [orange letter–denoted mutations (93, 94)]. (D) Photon-spike transduction mode arising in Volvox-derived C1V1-E83T: single red flash–single spike coupling (54) with moderately high speed/fidelity; later Volvox derivative bReaChES exhibits faster responses with ChETA modification for accelerated channel closure (77) (not shown). (E) Snapshot of most likely structure of the DC-pair region in C1C2 (blue lettering) and the C128T (Cys¹²⁸ → Thr) variant (red lettering) based on MD calculation (100) of restructured hydrogen-bonding network [yellow → blue dashed-line transition represents this SFO-mutation (52, 54) transition] and modified TM3-TM4 interaction (100), resulting in extension of open-state lifetime (–54) and many-orders-of-magnitude-increased light sensitivity of expressing cells (42, 52, 54). (F) Photon-spike transduction mode arising from C128T (SFO) mutation is bistable, ultra–light-sensitive, two-color switchable, and excitatory [note blue light actuation and green light termination (52, 54)]. (G) Photon-spike transduction mode arising from adding the Cys¹²⁸ SFO mutation (E) to Cl⁻-selective iC++ mutations (C) to create (64) SwiChR++ provides ultra–light sensitivity and is bistable, two-color switchable, and inhibitory under typical conditions (62, 64).

Fig. 5. Causal underpinnings of depression-related symptomatology identified via algal channel structure discovery and redesign

(A) Design of major opsin classes used together to identify brainwide dynamics of anhedonia (C1V1_TT/SSFO) (56): Model of red-shifted C1V1 RBP (red RSBH+; C1V1-specific side chains in cyan, based on 3UG9 structure) overlying modeled blue-responsive RBP (yellow RSBH+; green side chains, energy-optimized/calculated structure). Nearby DC-pair (SSFO) double mutant (D156A/C128S; new side chains in dark blue) shown (to illustrate relative positioning) that in the blue-responsive RBP confers stable activity shifts and blood oxygen level– dependent (BOLD) signal acquisition in MRI without heating or other artifacts. Expected further rearrangements and water influx are not shown. (B) Left: Tracks in rat brain for opsin injection and fiber-optic light access to mPFC (left arrow) and midbrain dopamine neurons (right arrow). Right: Testing causal influence of elevated mPFC activity (with blue-on/yellow-off SSFO) over communication within reward circuitry (from midbrain dopamine neurons controlled with C1V1). (C) Probing second-order brainwide dynamics. Natural prominent BOLD signal in dorsal and ventral striatum (left) recruited by dopamine neurons is potently suppressed (right) by mPFC excitability shift (56).