Chimeras of channelrhodopsin-1 and -2 from Chlamydomonas reinhardtii exhibit distinctive light-induced structural changes from channelrhodopsin-2

Abstract

Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.

Keywords: Channel Desensitization; Channelrhodopsin; Fourier Transform IR (FTIR); Gating; Ion Channel; Photobiology; Proton Transfer; Rhodopsin.

FIGURE 1.

Arrangement of the helices in chimeras of ChR1 and ChR2. A, schematic representation of the chimeric channel rhodopsins. The segments from ChR2 are colored yellow, and those from ChR1 are colored purple. Note that the amino acid sequences of TM3 in ChR1 and ChR2 (colored gray) are identical. A chimeric channelrhodopsin that has TM1 and TM2 from ChR1 and the other transmembrane regions from ChR2 is denoted ChR_2/5. Also, a chimera that has TM1 to TM5 from ChR1 and the other transmembrane regions from ChR1 is denoted ChR_5/2. B, the x-ray crystal structure of a chimeric channelrhodopsin called C1C2, which is nearly identical to ChR_5/2.

FIGURE 2.

Amino acid sequence alignment of ChR1, ChR2, and their chimeras (ChR_5/2, ChR_2/5, and C1C2). The amino acid sequences were aligned by ClustalW. The helix regions are shaded. The amino acid residues that are different between ChR1 and ChR2 are indicated in bold. The red residues in C1C2 are different from those in ChR_5/2. The residues that are substituted in this work are highlighted in yellow. Note that the amino acid sequence of TM3 is identical between ChR1 and ChR2 (black).

FIGURE 3.

Difference spectra between the photostationary state and the dark state of two chimeric channelrhodopsins, compared with that of ChR2. A, ChR_5/2. B, ChR_2/5. The difference spectra of ChR_5/2, ChR_2/5, and ChR2 are colored green, magenta, and orange, respectively. The double difference spectra were calculated by subtracting the light-induced difference spectrum of ChR2 with that of each chimera.

FIGURE 4.

Comparison of the light-induced difference spectra of two chimeric channelrhodopsins (ChR_5/2 and ChR_2/5) with that of ChR2 in the frequency region of the C=O stretching vibration of carboxyl group (1800–1670 cm⁻¹) and the symmetric COO⁻ stretching vibration (1450–1360 cm⁻¹). A, ChR_5/2 (green), ChR_2/5 (magenta), and ChR2 (orange). B, ChR2 (orange) and its E90Q mutant (red). C, ChR_5/2 (green) and its E129Q (red) and E136Q (blue) mutants. D, ChR_2/5 (magenta) and its E129Q (red) and E136Q (blue) mutants. E and F, the symmetric COO⁻ stretching region of C and D with the double difference spectra between the E129Q or E136Q mutant and the chimeras, respectively.

FIGURE 5.

Light-induced difference spectra of two chimeric channelrhodopsins (ChR_5/2 and ChR_2/5) containing mutations at the DC gate (Asp-195 and Cys-167) in the frequency region of the C=O stretching vibration of carboxyl group (1800–1670 cm⁻¹). A, ChR_5/2 (green) and its D195N mutant (blue). B, ChR_2/5 (magenta) and its D195N mutant (blue). C, ChR_5/2 (green) and its C167S mutant (red). D, ChR_2/5 (magenta) and its C167S mutant (red).

FIGURE 6.

Light-induced difference spectra of the D195N mutants of two chimeric channelrhodopsins compared with that of the corresponding D156N mutant of ChR2. A, ChR_5/2. B, ChR_2/5. The difference spectra of the Asp mutants of ChR_5/2, ChR_2/5, and ChR2 are colored green, magenta, and orange, respectively. The double difference spectra were calculated by subtracting the light-induced difference spectrum of ChR2 with that of each chimera.

FIGURE 7.

Light-induced difference spectra of the C167S mutants of two chimeric channelrhodopsins compared with that of corresponding ChR2 C128S mutant. A, ChR_5/2. B, ChR_2/5. The difference spectra of the Cys mutants of ChR_5/2, ChR_2/5, and ChR2 are colored green, magenta, and orange, respectively. The double difference spectra were calculated by subtracting the light-induced difference spectrum of ChR2 with that of each chimera.

FIGURE 8.

Time-resolved FTIR measurement of a ChR1/ChR2 chimera, C1C2, which is nearly identical to ChR_5/2. The intermediate spectra (blue) are reconstituted from decay-associated spectra obtained by global exponential fitting with three time constants: 92 μs (A), 177 μs (B), and 1.98 ms (C) as well as D (the spectral component at the infinite time).

FIGURE 9.

Upper panel, the spectral region of the carboxyl group C=O stretching vibration in the time-resolved FTIR spectra of a ChR1/ChR2 chimera, C1C2, which is virtually identical to ChR_5/2. The intermediate spectra are reconstituted from decay-associated spectra obtained by global exponential fitting with three time constants: 92 μs (A), 177 μs (B), and 1.98 ms (C), as well as D (the spectral component at the infinite time). These spectra are reproduced from Fig. 8. Lower panel, time courses of the absorption changes at 1759 cm⁻¹ (red), 1719 cm⁻¹ (green), and 1736 cm⁻¹ (blue) are shown with their global exponential fitting lines (thick lines, E).

FIGURE 10.

The x-ray crystal structure of C1C2. A, along the cation-conducting pathway. B, in the DC-gate region. Glu-129, Glu-136, Cys-167, and Asp-195, which were substituted in this study, correspond to Glu-90, Glu-97, Cys-128, and Asp-156 in ChR2, respectively. TM2, TM3, and TM4 from ChR1 are colored purple, whereas TM6 and TM7 from ChR2 are colored yellow. The all-trans retinal chromophore, which forms the Schiff base linkage with Lys-296, is rendered as a stick model.