Water-Containing Hydrogen-Bonding Network in the Active Center of Channelrhodopsin

Abstract

Channelrhodopsin (ChR) functions as a light-gated ion channel in Chlamydomonas reinhardtii. Passive transport of cations by ChR is fundamentally different from the active transport by light-driven ion pumps such as archaerhodopsin, bacteriorhodopsin, and halorhodopsin. These microbial rhodopsins are important tools for optogenetics, where ChR is used to activate neurons by light, while the ion pumps are used for neural silencing. Ion-transport functions by these rhodopsins strongly depend on the specific hydrogen-bonding networks containing water near the retinal chromophore. In this work, we measured protein-bound water molecules in a chimeric ChR protein of ChR1 (helices A to E) and ChR2 (helices F and G) of Chlamydomonas reinhardtii using low-temperature FTIR spectroscopy at 77 K. We found that the active center of ChR possesses more water molecules (9 water vibrations) than those of other microbial (2-6 water vibrations) and animal (6-8 water vibrations) rhodopsins. We conclude that the protonated retinal Schiff base interacts with the counterion (Glu162) directly, without the intervening water molecule found in proton-pumping microbial rhodopsins. The present FTIR results and the recent X-ray structure of ChR reveal a unique hydrogen-bonding network around the active center of this light-gated ion channel.

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

“…In the dark state of CaChR1, we estimate the O-H (O-D) frequency of the Hbonded O-H group of a dangling water at 3300 cm −1 (2450 cm −1 ), only slightly upshifted in respect to water W401 of BR and to the above discussed water molecule of the C1C2 chimera. 45 Thus, in homology with BR and C1C2, we suggest that this water molecule might take part, together with R166, E169, and D299 (see Fig. 8) and additional water molecules, in the H-bonding network of CaChR1 stabilizing the protonated SB.…”

“…45 Nevertheless, bands in the structurally sensitive amide I region are significantly different in the conductive states of CaChR1 (P 2 380 ) and CrChR2 (late P 2 390 and P 3 520 ), indicating differences in the structural changes that guide the opening of CaChR1 and CrChR2. We tentatively assigned bands in the amide I, II, and amide A regions of CaChR1 to changes in specific secondary structures.…”

“…8). 44 In an FT-IR study on the C1C2 chimera, a water molecule with the O-D stretch at 2378 cm −1 was assigned by its sensitivity to site-directed mutations to a water molecule H-bonded to D292 in the dark state, 45 a residue equivalent to D212 in BR. In the dark state of CaChR1, we estimate the O-H (O-D) frequency of the Hbonded O-H group of a dangling water at 3300 cm −1 (2450 cm −1 ), only slightly upshifted in respect to water W401 of BR and to the above discussed water molecule of the C1C2 chimera.…”

“…43,89 Nevertheless, FT-IR measurements at 77 K on the C1C2 chimera detected S-H difference bands between the dark state and the P 1 intermediate, tentatively assigned to C167. 45 Given their frequency, 2596(+)/2577(−) cm −1 , it was concluded that the S-H group is not H-bonded in the P 1 state but weakly H-bonded in the dark state. The latter result contradicts the X-ray structural model of the dark state C1C2 chimera.…”

“…44 Recently, the first report on active internal water molecules on a ChR was published, performed on the C1C2 chimera. 45 Light-induced FT-IR spectroscopy revealed changes in water O-D vibrations upon illumination at 77 K, i.e., between the closed dark and the early P 1 500 state. An unusually large number of active water molecules were identified as compared to other microbial rhodopsins.…”