Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Biol Chem
2012 Feb 24;2879:6904-11. doi: 10.1074/jbc.M111.327700.
Show Gene links
Show Anatomy links
In channelrhodopsin-2 Glu-90 is crucial for ion selectivity and is deprotonated during the photocycle.
Eisenhauer K
,
Kuhne J
,
Ritter E
,
Berndt A
,
Wolf S
,
Freier E
,
Bartl F
,
Hegemann P
,
Gerwert K
.
???displayArticle.abstract???
The light-activated microbial ion channel channelrhodopsin-2 (ChR2) is a powerful tool to study cellular processes with high spatiotemporal resolution in the emerging field of optogenetics. To customize the channel properties for optogenetic experiments, a detailed understanding of its molecular reaction mechanism is essential. Here, Glu-90, a key residue involved in the gating and selectivity mechanism of the ion channel is characterized in detail. The deprotonation of Glu-90 during the photocycle is elucidated by time-resolved FTIR spectroscopy, which seems to be part of the opening mechanism of the conductive pore. Furthermore, Glu-90 is crucial to ion selectivity as also revealed by mutation of this residue combined with voltage clamp experiments. By dynamic homology modeling, we further hypothesized that the conductive pore is flanked by Glu-90 and located between helices A, B, C, and G.
FIGURE 1. P480 to D470 relaxation rates of ChR2 WT and E90Q. Amplitude spectra of the relaxation rate from P480 to D470 of WT ChR2 (black) and the mutant E90Q (red) are shown. The negative carbonyl band of Glu-90 at 1718 cm−1 and the corresponding positive carboxylate band, either at 1381 cm−1 or 1439 cm−1, are missing in the mutant. The insets show the subtraction results of the WT and E90Q spectra. The C=O (1718 cm−1, red stripes) of Glu-90 and its corresponding COO− band (1381 cm−1 or 1439 cm−1, red stripes) disappear in the mutant. Therefore, these bands can be unambiguously assigned to Glu-90 deprotonation. In Gln-90, which has a fixed protonation state, a difference band at 1688/1678 cm−1 (blue stripes) is observed, which indicates its H-bond or environmental change.
FIGURE 2. Time-resolved absorbance changes in the WT protein induced by 20 ns laser excitation. UV-visible (A) and FTIR (B) spectra are shown. The amplitude spectra (orange and red) were generated by a global fit of the complete combined dataset. In A: a P520-D470 difference spectrum (black) averaged over the first 10 ms is shown. The red shift of the absorption maximum indicates the accumulation of the P520 intermediate. In the corresponding IR difference spectrum, now assigned to the P520 intermediate, the deprotonation of Glu-90, indicated by the negative band at 1718 cm−1, is observed. The first observed rate of 30 ms represents the P520 to P480 transition (orange). Here, the band shift of a carboxylic residue from 1728 to 1737 cm−1 is monitored. The slowest rate (red) of 45 s represents the P480 to D470 transition. The negative band at 1718 cm−1 indicates the reprotonation of Glu-90. Individual time-resolved absorbance changes at 520 nm in C and at 1718 cm−1 in D (Glu-90) are shown. The time-resolved data confirm that Glu-90 is already deprotonated in P520, being the putative open state. Whether deprotonation takes place in an earlier transition or simultaneously with the appearance of the P520 intermediate cannot be distinguished yet. Furthermore, the absorbance changes at 1662 cm−1 (E) show that the large structural changes (not time-resolved) of the protein backbone relax in two steps, in the P520 to P480 and the P480 to D470 transitions.
FIGURE 3. Electrophysiological characterization of ChR2-WT and mutants of Glu-90. Photocurrents of ChR2-WT (A) at pHo 7.5 and pH 4 and E90H (B) at pHo 7.5 in the absence and presence of 100 mm Na+ at −100 mV (blue bar: 470 nm excitation). NMG-Cl was substituted by NaCl. C, averaged initial photocurrents (I0) at pHo 7.5 and pHo 4 at −100 mV. Significance: p < 0.05 (*), p < 0.005 (***). Current-voltage relation of the initial current (I0) at 100 mm Na+ for wild-type ChR2 (D) and the mutants E90Q, E90A, E90H, and E90K (E–H). Measurements at pHo 7.5 and −100 mV were used as reference currents (Iref); error bars: ±S.E.
FIGURE 4. Water densities (43) reflecting the protein-internal volume in the ChR2 homology model occupied by water molecules during MD simulations depending on the protonation state of Glu-90. Water density distribution changes within ChR2 due to deprotonation of Glu-90. When Glu-90 is protonated (left panel), two distinct regions of intruding water molecules are observed, with Glu-90 being found in the middle of the barrier between them. When Glu-90 is deprotonated (right panel), the water influx is altered. The former unsolvated region around Glu-90 starts to fill up with water. The extracellular side is still more solvated than the intracellular side of the protein. The alteration of water densities results in an almost continuously spanning water-filled pore.
FIGURE 5. Putative location of the conductive pore within the helical bundle of the BR-based ChR2 homology model (intracellular view). ChR2 is shown in gray, the chromophore retinal is highlighted in yellow, Glu-90 is in purple, and penetrating water molecules are shown as blue surfaces. MD simulation reveals that the location of the conductive pore within the helical bundle of ChR2 is between helices A, B, C, and G, in direct contact with the chromophore and the RSB, indicated by the red circle. MD simulations reveal Glu-90 to be flanking the conductive pore.
Bamann,
Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond.
2010, Pubmed,
Xenbase
Bamann,
Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond.
2010,
Pubmed
,
Xenbase
Belrhali,
Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution.
1999,
Pubmed
Berndt,
High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels.
2011,
Pubmed
Berndt,
Bi-stable neural state switches.
2009,
Pubmed
,
Xenbase
Canutescu,
A graph-theory algorithm for rapid protein side-chain prediction.
2003,
Pubmed
Doszczak,
Prediction of perception: probing the hOR17-4 olfactory receptor model with silicon analogues of bourgeonal and lilial.
2007,
Pubmed
Ernst,
Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits.
2000,
Pubmed
Ernst,
Photoactivation of channelrhodopsin.
2008,
Pubmed
,
Xenbase
Freier,
Proton transfer via a transient linear water-molecule chain in a membrane protein.
2011,
Pubmed
Fritze,
Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation.
2003,
Pubmed
Garczarek,
Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy.
2006,
Pubmed
Gelis,
Prediction of a ligand-binding niche within a human olfactory receptor by combining site-directed mutagenesis with dynamic homology modeling.
2012,
Pubmed
Gerwert,
Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.
1990,
Pubmed
Gerwert,
Role of aspartate-96 in proton translocation by bacteriorhodopsin.
1989,
Pubmed
Govorunova,
New channelrhodopsin with a red-shifted spectrum and rapid kinetics from Mesostigma viride.
2011,
Pubmed
Gradmann,
Rectification of the channelrhodopsin early conductance.
2011,
Pubmed
,
Xenbase
Gunaydin,
Ultrafast optogenetic control.
2010,
Pubmed
,
Xenbase
Hess,
GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation.
2008,
Pubmed
Joh,
Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins.
2008,
Pubmed
Kandt,
Dynamics of water molecules in the bacteriorhodopsin trimer in explicit lipid/water environment.
2004,
Pubmed
Kufareva,
Status of GPCR modeling and docking as reflected by community-wide GPCR Dock 2010 assessment.
2011,
Pubmed
Lanyi,
Proton transfers in the bacteriorhodopsin photocycle.
2006,
Pubmed
Lin,
Characterization of engineered channelrhodopsin variants with improved properties and kinetics.
2009,
Pubmed
,
Xenbase
Marin,
The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction.
2000,
Pubmed
Molday,
Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes.
1983,
Pubmed
,
Xenbase
Müller,
Projection structure of channelrhodopsin-2 at 6 Å resolution by electron crystallography.
2011,
Pubmed
Nagel,
Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.
2003,
Pubmed
,
Xenbase
Radu,
Conformational changes of channelrhodopsin-2.
2009,
Pubmed
Ritter,
Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy.
2008,
Pubmed
Ruffert,
Glutamate residue 90 in the predicted transmembrane domain 2 is crucial for cation flux through channelrhodopsin 2.
2011,
Pubmed
,
Xenbase
Schneider,
The structure of active opsin as a basis for identification of GPCR agonists by dynamic homology modelling and virtual screening assays.
2011,
Pubmed
Schoenenberger,
Temporal control of immediate early gene induction by light.
2009,
Pubmed
Sineshchekov,
Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii.
2002,
Pubmed
Stehfest,
The branched photocycle of the slow-cycling channelrhodopsin-2 mutant C128T.
2010,
Pubmed
Stehfest,
Evolution of the channelrhodopsin photocycle model.
2010,
Pubmed
Sugiyama,
Photocurrent attenuation by a single polar-to-nonpolar point mutation of channelrhodopsin-2.
2009,
Pubmed
Suzuki,
Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization.
2003,
Pubmed
Tieleman,
Lipid properties and the orientation of aromatic residues in OmpF, influenza M2, and alamethicin systems: molecular dynamics simulations.
1998,
Pubmed
Verhoefen,
The photocycle of channelrhodopsin-2: ultrafast reaction dynamics and subsequent reaction steps.
2010,
Pubmed
Wolf,
Directional proton transfer in membrane proteins achieved through protonated protein-bound water molecules: a proton diode.
2010,
Pubmed
Wolf,
Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand binding site.
2008,
Pubmed
Yizhar,
Optogenetics in neural systems.
2011,
Pubmed