Channelrhodopsin-1 initiates phototaxis and photophobic responses in chlamydomonas by immediate light-induced depolarization

Abstract

Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H+-channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H+, CHR1 also conducts Na+, K+, and Ca2+. The kinetic selectivity analysis demonstrates that H+ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1505 and CHR1463, that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.

Figure 1.

Downregulation of ChR1 in the Chlamydomonas Strain CW2. (A) A representative immunoblot analysis of three RNAi mutants, H17, H77, and H84, and of the untransformed CW2 recipient strain (n = 3). Vegetative cells (veg) and gametes (gam) were analyzed with anti-CHR1 antibody or anti-CHR2 antibody. (B) Schematic of the pPB-ASCHR1 plasmid used for Chlamydomonas transformation, containing the hygromycin selection marker (aph7′) (Berthold et al., 2002) driven by the Chlamydomonas β-tubulin promotor (P_Tub) and the RNAi hairpin construct (COP3 genomic DNA, 1538 to 3190 bp, and cDNA, 471 to 972 bp in antisense direction) under control of the Hsp79/RbcS2-promotor (P_AR). (C) Immunofluorescence of vegetative cells of the wild-type strain CC124 and gametes of CW2 and the RNAi-mutant H17. CHR1 protein is visualized in red, and α-tubulin is shown in green. Bars = 10 μm. (D) Photoreceptor currents measured from gametes of the recipient strain CW2 and the CHR1 antisense transformants H84, H77, and H17 under the “eyespot in” configuration. Cells were stimulated with a green light pulse (λ = 510 ± 20 nm, 3·10²² photons m⁻²s⁻¹). I_p and I_ps are the fast and slow photoreceptor currents, respectively. (E) Relation between CHR1 content and average I_p amplitude in CW2 and antisense transformants. CHR1 content of CW2 gametes was set at 100%. Protein content and currents were determined from the same culture. Each current amplitude represents an average of five measurements from nine individual cells including se. (F) Phototaxis (dish test) of CW2 and H17 gametes. Cells were exposed to white light (∼7 W/m²), and cell migration was observed for 6 min. (G) Swimming speed of CW2 and H17 gametes and photophobic responses to flashes of green light (arrows; 10 μs, λ = 510 ± 40 nm at 3·10²² photons m⁻²s⁻¹). (H) and (I) Phototactic orientation of CW2 (H) and H17 (I) vegetative parallel cultures as monitored in a light scattering apparatus during light pulses of 455 ± 20 nm at six different light intensities, numbered 1 to 6 (6.0·10¹⁷, 1.2·10¹⁸, 2.4·10¹⁸, 6.0·10¹⁸, 1.2·10¹⁹, and 6.0·10¹⁹ photons m⁻²s⁻¹, respectively). The traces are averages of three individual measurements from one out of two similar independent experiments. The CHR1 and CHR2 content at the time of the measurement may be estimated from the protein blots (insets). The drawing at the bottom explains the orientation pattern of the culture during the experiment. (J) The signal amplitudes of CW2 and H17 vegetative cells after 4 s of exposure to three characteristic wavelengths of light (455, 505, and 530 ± 20 nm) plotted versus the photon irradiance. (K) Phototactic orientation of CW2 and H17 gametes (parallel cultures) as monitored in a light scattering apparatus during light pulses of 505 ± 20 nm and at different light intensities. (L) The signal amplitudes measured in CW2 and H17 gametes after 4 s in the light plotted against the photon irradiance.

Figure 2.

Photocurrents and Phototaxis at pH_o 9. (A) Flash-induced photocurrent I_P and the subsequent flagellar current I_F of CW2 gametes at pH_o 6.8 and 9 in the “eyespot in” configuration. Cells were stimulated with flashes of 510 ± 40 nm for a 10-μs duration (1, 1%; 2, 2.5%; 3, 6%; 4, 10%; 5, 25%; 6, 50%; 7, 100%, corresponding to 2.2·10¹⁹ photons/m²). pH_o in the bath solution was kept at 6.8 as indicated in the diagram. (B) Initial phototactic activity of CW2 and H17 gametes at pH_o 6.8 and 9 upon stimulation with a short light pulse of 510 ± 20 nm (1·10¹⁸ photons m⁻² s⁻¹). A signal of 100 mV·s⁻¹ corresponds to a phototactic movement of ∼100 μm s⁻¹. Note that this assay only allows reliable recording of the cell movement during a few seconds after the light is switched on (Uhl and Hegemann, 1990).

Figure 3.

Cation Dependence of the CHR1 Photocurrents. CHR1 was expressed in Xenopus oocytes to measure light-induced currents, using the two-electrode voltage-clamp technique. (A) Photocurrents recorded at pH_o 6.0. Inward photocurrents were observed upon illumination (λ = 500 ± 40 nm, shown as a bar) in the presence (top) and absence (bottom) of 100 mM Na⁺ in the bath solution. The membrane potential was held at −125 mV. The traces shown are recorded from the same oocytes (n = 6). (B) Photocurrents recorded at pH_o 9.0. In the presence of 100 mM Na⁺ in the bath solution (top), inward currents were observed upon illumination, whereas almost no current was observed in the absence of Na⁺ (bottom). Currents were also observed when Na⁺ was exchanged for 70 mM Ca²⁺ (middle). The traces shown are recorded from the same oocyte (n = 5). Experiments were performed at pH_o 9.0, where H⁺ inward currents are negligible. The membrane potential was held at −125 mV. (C) Current ratios for different cations. Inward photocurrents at −100 mV were normalized to those measured at 100 mM Na⁺, pH_o 6. Photocurrents were larger at lower pHo due to the contribution of H⁺ permeation. Net H⁺ permeation is estimated from the measurement with 100 mM NMG-Cl. Note that significant Ca²⁺ conductance was observed at pH_o 9.0. (D) [Na⁺] dependence of the CHR1 photocurrent. I/V plot of the photocurrent at different [Na⁺]. Bath solution contained 0.1 mM CaCl₂, 0.1 mM MgCl₂, 5 mM glycine-NMG pH_o 9, plus variable concentrations of NaCl as indicated, and NMG-Cl, up to a total cation concentration of 105 mM. (E) [Ca²⁺] dependence of CHR1 photocurrent. I/V plot of photocurrent at different [Ca²⁺]. The solutions contained 0.1 mM MgCl₂ and 5 mM glycine-NMG, pH_o 9, plus variable CaCl₂ concentrations and NMG-Cl up to a total cation concentration of 105 mM.

Figure 4.

Kinetic Selectivity Analysis. (A) Minimum reaction scheme and nomenclature for competitive translocation of H⁺ and an alternate monovalent cation, here K⁺, through an ion translocating enzyme (E) according to Gradmann et al. (1987). Voltage-sensitive and temporally resolved reactions between inside and outside can be determined explicitly by steady state current–voltage relationships recorded at different external concentrations of H⁺ and K⁺ and are represented by standard line width. Bold lines: pK_H = ln((1/K₂) and pK_K = ln((1/K₄) mark fast binding and debinding reactions outside with the equilibrium constants K₂ = k₃₂/k₂₃ and K₄ = k₃₄/k₄₃ for fast binding and debinding reactions outside, correspond to pK_H = -ln₁₀(1/K₂) and pK_K = -ln₁₀(1/K₄), respectively. z_E, apparent charge number of unoccupied binding site (Andersen, 1989). Gray, supplementation of reaction scheme by distinction of fast inner binding equilibria and slow translocation steps, which could be identified when inner concentrations of H⁺ and K⁺ were changed as well during experimentation (not done here). (B) Example of analysis by fitting the independent parameters of the model in (A) to experimental steady state current–voltage relationships (points) in the presence and absence of 100 mM K⁺ at pH_o 9 and pH_o 6. Inset table: numerical results of fit (value) and sensitivity (sens.); sensitivity is expressed as increase of mean sd in 0.1% upon ±10% deviations of the parameter from its fitted optimum value.

Figure 5.

CHR1 Spectra. (A) and (B) Spectra of purified dark-adapted CHR1 in dodecyl maltoside solution in darkness ([A]; dark) and upon permanent illumination with blue light from a 470-nm LED ([B]; light) at different pH values. Inset shows overlay of spectra at pH 4.5 and 8.0, respectively. Bottom panels are enlarged views of the top panels. (C) Typical recording traces of CHR1 expressed in a Xenopus oocyte. CHR1 was excited with 10-ns laser flashes of various wavelengths (indicated by an arrow). The membrane voltage was clamped at −75 mV. (D) Action spectra resulting from stimulation with laser flashes (as in [B]) at pH_o 9.0, 7.5, 6.5, and 5.5. Peak wavelengths are indicated by arrows. Data points are averages of 20 to 25 recordings. The current amplitudes were compared for equal photon irradiance (photons m⁻²).