Structural Model of Channelrhodopsin

et al. 2015

Biophysical Journal

Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H2O and in D2O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm(-1) to 1738 cm(-1) in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm(-1) in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm(-1) and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.

Section: Discussionmentioning

confidence: 99%

Kinetic and Vibrational Isotope Effects of Proton Transfer Reactions in Channelrhodopsin-2

Resler

Schultz

et al. 2015

Biophysical Journal

“…An acidic residue at position 253 but not at position 123 is conserved in all ChRs sequenced so far (40). Whereas the replacement of D253 by nonprotonatable residues leads to the loss of channel function [D253A in C1C2 (6) and D253N in ChR2; SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 99%

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating

Proc. Natl. Acad. Sci. U.S.A.

Resler

Krause

et al. 2013

162

414

The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P 2 390 and P 3 520 ) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t 1/2 = 60 μs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t 1/2 = 10 μs), with the latter being reprotonated by aspartic acid 156 (t 1/2 = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P 4 480 state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel.O ptogenetics provides new tools to neurophysiologists to steer cellular responses with unprecedented temporal and spatial resolution. The former takes advantage of light as an ultrashort trigger, whereas the latter is achieved by genetically encoding and directing photosensitive proteins to specific cell types. The most prominent among the optogenetics tools is channelrhodopsin (ChR), which was found to be the first light-gated ion channel of its kind (1, 2). This discovery paved the way for an exponentially growing number of neurophysiological applications, ranging from single cells to living animals (3). Light-gated ion permeation by ChR expands the various modes of action of the large family of microbial rhodopsins already comprising light-driven ion pumps and sensors (4). Among the various ChRs, which differ mostly in cation selectivity (3), ChR2 is used in the majority of optogenetic applications because of the higher expression yield in mammalian cells.A projection structure of the heptahelical ChR2 showed a dimer with the contact interface between helices C and D suggested to form the cation channel (5). More recently, a chimeric ChR (C1C2) was constructed by linking the last two helices (F and G) of ChR2 to the first five (A to E) of ChR1 and resolved by X-ray crystallography to 2.3 Å (6). The high-resolution structure confirmed the dimeric arrangement and identified an electronegative extracellular pore in each monomer framed by helices A, B, C, and G. Accompanying electrophysiological experiments on point mutants indicated r...

“…71 This experimental observation challenges the conclusions from an homology model of CrChR2 refined by QM/MM simulations, suggesting that cysteine residues showing notable vibrational changes in the S-H stretch could be H-bonded only at the sulfur atom. 74 Therefore, the observed 24 cm −1 downshift of the S-H band between the dark state and the P 2 380 state of CaChR1 must originate from an increase of the H-bond strength of the S-H group as a H-bond donor: from moderately to strongly H-bonded.…”

Section: F Hydrogen-bonding Changes Of the Thiol Group Of Cysteine Rmentioning

confidence: 99%

“…74,87 Consequently, a change in H-bonding strength of a cysteine side chain might originate either by a change in the pitch of the helix in the case of an intrahelical H-bond or a change in the relative orientation of two helices in the case of an interhelical H-bond. For CrChR2, it has been shown that the photocycle is associated with the movement of transmembrane helices, predominantly of helices B and F. 88 Because helix F of CaChR1 lacks cysteine residues, the cysteine residues in helix B (C133, C134, and C141) are prime candidates to undergo a measurable change in their S-H stretch frequency as a result of the structural changes.…”

Section: Hydrogen-bonding Changes In the Side Chain Of Internal Cymentioning

confidence: 99%

“…84 The first cysteine, C137, is conserved in CrChR1 (C222) and CrChR2 (C183), and in the derived C1C2 chimera (C222). 44,74 The above cysteine is not conserved in CaChR1 (T229), but two cysteine residues are present nearby in helix E (C231 and C232). Thus, these two residues are prime candidates to change their S-H stretches during the photocycle of CaChR1, as well.…”

Section: Hydrogen-bonding Changes In the Side Chain Of Internal Cymentioning

confidence: 99%

See 1 more Smart Citation

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

The Journal of Chemical Physics

Muders

Schlesinger

et al. 2014

Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the ( −1 ) to moderately strongly (3300 cm −1 ) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the "dangling" water molecules within the protein, providing a better understanding of their functional relevance in CaChR1.