Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M708039200 on November 9, 2007

J. Biol. Chem., Vol. 283, Issue 3, 1637-1643, January 18, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/3/1637    most recent
M708039200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ernst, O. P.
Right arrow Articles by Hegemann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ernst, O. P.
Right arrow Articles by Hegemann, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Photoactivation of Channelrhodopsin*Formula

Oliver P. Ernst{ddagger}1, Pedro A. Sánchez Murcia{ddagger}2, Peter Daldrop§, Satoshi P. Tsunoda§, Suneel Kateriya, and Peter Hegemann§3

From the {ddagger}Institut für medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany, the §Institut für Biologie, Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany, and the Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

Received for publication, September 26, 2007 , and in revised form, November 6, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. In optogenetics, ChRs are widely applied for light-triggering action potentials in cells, tissues, and living animals, yet the spectral properties and photocycle of ChR remain obscure. In this study, we cloned a ChR from the colonial alga Volvox carteri, VChR. After electrophysiological characterization in Xenopus oocytes, VChR was expressed in COS-1 cells and purified. Time-resolved UV-visible spectroscopy revealed a pH-dependent equilibrium of two dark species, D470/D480. Laser flashes converted both with {tau} {approx} 200 µs into major photointermediates P510/P530, which reverted back to the dark states with {tau} {approx} 15-100 ms. Both intermediates were assigned to conducting states. Three early intermediates P500/P515 and P390 were detected on a ns to µs time scale. The spectroscopic and electrical data were unified in a photocycle model. The functional expression of VChR we report here paves the way toward a broader structure/function analysis of the recently identified class of light-gated ion channels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The behavior of microalgae like Chlamydomonas is controlled by primordial eyes. The earliest events that are recorded from these eyes after light excitation are extremely fast photoreceptor currents (1-3) that are carried by Ca2+ and H+ under most conditions (3, 4). The currents are generated by channel-rhodopsins (ChRs),4 microbial type rhodopsins with intrinsic ion conductance (5-7). ChRs gain their light sensitivity and gating properties from the covalently bound chromophore all-trans-retinal, which isomerizes after light absorption (8, 9). Studies in Xenopus oocytes and HEK293 cells revealed ChRs to be proton channels, which also conduct cations like Na+, K+, and Ca2+ (5, 6, 10). In these studies, light flashes evoked the ion conductance with a time constant of 200 µs after the flash, thus closely resembling the photocurrent rise in living algae (2, 6).

The observation that ChRs rapidly depolarize membranes within a few milliseconds in response to brief light flashes motivated neurophysiologists to use ChRs for controlling nerve cell firing. Originally, three groups in parallel expressed ChR in hippocampal neurons and triggered action potentials by short light pulses. The neurons followed the light stimulation protocols with action potential firing up to 30 Hz (10-12). ChR was also used to control neuronal activity in living animals, such as chicken embryos (11), Caenorhabditis elegans (13, 14), Drosophila (15), and mouse (16). In all cases, action potentials could be evoked by application of blue light to cells that were naturally light insensitive. The unique properties of ChR opened a new field of optogenetic technology for neuroscience and medical applications (17-19). However, despite the significant efforts that were made to make ChR available for such goals (20), the structure/function relationships of ChR, including the photoreactions as well as the ion channeling mechanism, remain unknown.

Here we describe the cloning and functional characterization of a ChR from the colonial alga Volvox carteri (VChR; see Fig. 1A). First, VChR was expressed in Xenopus oocytes and confirmed as a light-gated cation channel. Subsequently, it was expressed in COS-1 cells and functionally purified in detergent. This allowed us to obtain time-resolved UV-visible spectroscopic data and gain insight into the spectral properties and photocycle of this protein class. We identified two dark states and three photocycle intermediates with pH-dependent variations. Our studies on the transitions between the different states under various pH and light conditions are interpreted in a model that unifies the photocurrent kinetics and spectroscopic data.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Cloning of the VChop DNA—Nucleotide sequences homologous to channelopsins Chop1 and Chop2 from Chlamydomonas reinhardtii were searched in a preliminary genome data base of V. carteri that is available within the Chlamydomonas genome project (genome.jgi-psf.org). Chop gene products are opsin apoproteins that form ChRs when reconstituted with the chromophore all-trans-retinal. Using the sequence information from the BLAST search, a 1797-bp fragment of the VChop cDNA was generated by reverse transcription-PCR from RNA isolated from a young V. carteri culture. See the supplemental data for details of cloning and sequence information on the resulting VChop cDNA fragment (GenBankTM accession number DQ094781).

Heterologous Expression in Xenopus Oocyte—A VChop cRNA fragment encoding VChR1-307 (amino acids 1-307) was synthesized in vitro from NheI-linearized pGEMHE-plasmid (5, 6) by using T7 RNA polymerase (mMessage mMachine, Ambion). Oocytes prepared from female Xenopus (5, 6) were obtained from Ecocyte Bioscience and were injected with 50 nl of VChop cRNA fragment (0.5-1.0 µg/µl) and incubated in the dark at 18 °C in Ringer solution (96 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM MOPS, pH 7.5) in the presence of 1 mg/ml penicillin, 1 mg/ml streptomycin, 0.5 mM theophylline, and 1 µM all-trans-retinal (Sigma).

Electrophysiology—Two-electrode voltage clamp measurements on Xenopus oocytes were performed 3-7 days after injection at 22 °C under the conditions described before (21, 22). For high resolution experiments a Turbo Tec-05X amplifier (NPI Electronic, Tamm, Germany) was used. The data obtained were normally the averages of three measurements. The resistances of microelectrodes were 0.5-1.5 M{Omega}. For recording of fast kinetics, 10-ns flashes (400-600 nm) from a Rainbow OPO (OPOTEK, Carlsbad, CA) pumped by the third harmonic of a Brilliant b Nd-YAG-Laser (Quantel, Les Ulis Cedex, France) were applied via a 1-mm light guide. The current amplifier Tec-05X was compensated so that the voltage change was kept below 1 mV at a half-saturating laser flash. The data were recorded at high gain with a sampling rate of 250 kHz. The laser intensity varied from flash to flash within a range of 5%. Bath solution was 100 mM NaCl, 0.1 mM CaCl2, 0.1 mM MgCl2 with 5 mM MOPS-NaOH, pH 7.5, or 5 mM citrate, pH 4.0. The conditions for light pulse experiments were 500 ± 5 nm, 2 x 1022 photons m-2 s-1.

VChR Expression in COS-1 Cells—For expression in COS-1 cells (ATCC, CRL-1650), a human codon-optimized synthetic VChop DNA fragment (corresponding to amino acids 1-307 of the native protein, accession number DQ094781), plus the C-terminal ETSQVAPA sequence (1D4 epitope (23)) was designed and purchased from GeneArt (Regensburg, Germany). The synthetic VChop DNA was inserted between the EcoRI-NotI sites of the expression vector pMT3 (24). Tissue culture, transient transfection with the resulting VChR-pMT3 vector, reconstitution with chromophore, and subsequent purification of VChR was performed basically as described for bovine rhodopsin (25, 26). Three days after transfection, the cells were harvested and reconstituted with all-trans-retinal (final concentration, 1.5 µM). VChR was solubilized with dodecyl maltoside and purified by immunoaffinity adsorption using rho 1D4 antibody coupled to CNBr-activated Sepharose 4 Fast Flow (GE Healthcare). VChR was eluted with 100 µM of an 18-mer peptide corresponding to the C-terminal rhodopsin sequence in 0.03% (w/v) dodecyl maltoside, 10 mM 1,3-bis-[tris(hydroxymethyl)methylamino] propane, pH 6.0 (25, 26). Eluates were dialyzed and/or concentrated using Centricon YM-10 (Millipore) concentrators and stored at 4 °C or -40 °C. The mutant VChR-K252G was generated by site-directed mutagenesis (QuikChange kit; Stratagene). The mouse anti-rhodopsin monoclonal antibody rho 1D4 used for purification and Western blotting of VChR was purchased from the University of British Columbia (Dr. R. Molday). Purified VChR was deglycosylated with peptide-N-glycosidase F (Roche) according to the manufacturer's instructions.

Spectroscopy—For absorption spectroscopy and slow kinetics at 20 °C, a Cary 50 Bio spectrophotometer (Varian Inc.) with a spectral resolution of 2 nm was used. The samples were illuminated for 10 s with a blue Luxeon LED (Philips Lumileds, San Jose, CA) with a wavelength of 456 nm, 90 milliwatt cm-2, 2 x 1021 photons m-2 s-1. Transient spectroscopy was performed on a LKS.60 flash photolysis system (Applied Photophysics Ltd., Leatherhead, UK) at 22 °C. Excitation pulses (10 ns, 450 nm) were provided by a tunable Rainbow OPO/Nd-YAG-Laser laser system (see above). Laser energy was adjusted to 3.5 ± 0.16 mJ/shot. The instrument used a Xe-Lamp (150 W) as a monitoring light source, which was pulsed during short time experiments. Monochromators before and after the sample were set to spectral resolutions of 18.6 and 9.3 nm, respectively. For detection, a 1P28 photomultipier (Hamamatsu Photonics) was used, and the signal was recorded with an Infinium Oscilloscope (Agilent Technologies). 32,000 data points were recorded in each measurement and compressed by LKS.60 software to files of 500 data points. 50 of these points were recorded before and 450 after laser excitation. To avoid artifacts and scatter, only data points 60-500 were used for analysis. Data analysis was performed with Matlab 7.01 (The MathWorks, Natick, MA). Singular value decomposition of representative data sets was performed to identify significant components that were used for reconstruction of the three-dimensional spectra (see supplemental data). For measurements at pH 6, freshly prepared VChop was used. For experiments at pH 7.3 and 4.5, freshly prepared VChop was dialyzed and/or diluted with buffer containing 0.03% dodecyl maltoside, yielding 100 mM NaCl, 0.1 mM CaCl2, 0.1 mM MgCl2 with 10 mM MOPS, pH 7.3, or 10 mM citrate, pH 4.5, and the pH was measured thereafter. pH titration was performed with 20 mM buffer (citrate, pH 4.5-6; MOPS, pH 7; or Tris, pH 8) in 100 mM NaCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.03% dodecyl maltoside.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Functional Studies on VChR in Xenopus Oocytes—In the past, ChRs from Chlamydomonas were difficult to express in standard expression systems like Escherichia coli, yeast, or COS cells (27). We therefore isolated a ChR-related cDNA from young V. carteri colonies (Fig. 1A). The cDNA encoded a large Volvox-ChR protein, VChR (599 amino acids), with seven transmembrane helices (TM) and a long C-terminal extension. We only studied the 7TM region of VChR (Fig. 1B) corresponding to amino acids 1-307 (see supplemental data) because earlier work on Chlamydomonas ChR1 and ChR2 (5, 6) showed that the long C-terminal extension had little influence on the ion conducting properties. In VChR-expressing Xenopus oocytes, light pulses evoked an initial transient current that relaxed toward a stationary plateau (steady state; {tau}1 = 40-85 ms depending on the extracellular H+ concentration (pHo) and membrane voltage (Fig. 2A). The current increased with progressing negative voltage and with decreasing pHo (Fig. 2, A and B), whereas the transient fraction of the current was particularly dominant at high pHo and low negative or positive voltage values. After the light is off, the current decays biexponentially with {tau}2 = 15-25 ms and {tau}3 = 60-140 ms (-100 mV, pHo 4-7.5).


Figure 1
View larger version (55K):
[in this window]
[in a new window]

  		FIGURE 1.
V. carteri. A, Volvox colony with a few thousand small Chlamydomonas-like somatic cells and 16 reproductive gonidia (courtesy of Armin Hallmann). The inset shows a few enlarged somatic cells with their red eyes. The cells at the surface of the colony with their flagella are shown schematically. B, VChR in the eye spot is a light-gated cation channel, which is composed of a seven transmembrane helix domain with the all-trans-retinal chromophore and a C-terminal tail of unknown function. Carotenoid-rich vesicles reflect the light and create a front to back contrast at the location of the ChR-photoreceptor. Absorption of light by VChR induces opening of the channel and thus changes of the membrane voltage.

 


Figure 2
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 2.
Recordings of two-electrode voltage clamp from VChR-expressing Xenopus oocytes. Voltage and light dependence of the VChR photoreceptor currents. A, photocurrents on VChR-expressing Xenopus oocytes measured with two-electrode voltage clamp in response to light pulses (indicated by a gray bar) ({lambda} = 500 nm, 2 x 1022 photons m-2 s-1) at seven different holding potentials. {tau}1-{tau}3 are time constants of the current decay as indicated. Bath solution contained 100 mM NaCl, 0.1 mM MgCl2, 0.1 mM CaCl2, and 5 mM MOPS-NaOH, pH 7.5. B, photocurrent/voltage (I/V) plot of the two photocurrent components; transient fraction (squares) and steady state fraction (circles). The data are averages from three independent experiments (filled symbols, pHo 7.5; open symbols, pHo 6.0). pHo was adjusted to 6.0 with 5 mM citrate. C, photocurrents in response to various light intensities as indicated. 100% corresponds to 1.6 x 1022 photons m-2 s-1. Bath solution was the same as in A. The membrane voltage was clamped at-100 mV. D, the (total) peak amplitude and the stationary level (at the end of the light pulse) of the photocurrents as shown in C are plotted as a function of light intensity (average of three experiments).

 


Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		FIGURE 3.
Dependence of photocurrent on double light pulse stimulation, wavelength, and ionic conditions. Two-electrode voltage clamp recordings from VChR-expressing Xenopus oocytes. A, two light pulses indicated as gray bars with 1-s dark interval evoke transient peak currents of different size but lead to similar stationary currents. B, almost full recovery of the second transient peak current is observed after a 30-s dark period. Bath solution contained 100 mM NaCl, 0.1 mM MgCl2, 0.1 mM CaCl2, and 5 mM MOPS-NaOH, pH 7.5. Membrane voltage was clamped at 0 mV. C, recovery of the second transient peak current (I2) in relation to the first transient peak current (I1) in double light pulse experiments as shown in A and B. D, photocurrent traces obtained by stimulation with 10-ns laser flashes ({lambda} = 470 nm, membrane potential as indicated). E, action spectra resulting from stimulation with 10-ns laser flashes of various wavelengths at pHo 7.5 and pHo 4.0, peaking at 470 and 484 nm, respectively. The data points shown are averages of 20-25 recordings. F, cation selectivity measured under three different pHo conditions with membrane potential at -100 mV. Bath solution contained 100 mM LiCl, NaCl, KCl or NMG-Cl with 0.1 mM CaCl2, and 0.1 mM MgCl2 plus 5 mM citrate (for pH 4.0 and 6.0) or 5 mM MOPS-NaOH (for pH 7.5). The currents were normalized to those recorded with NaCl and pHo 4. The data shown are the averages of more than three independent experiments.

 
The current amplitude certainly also depends on the light intensity, but the stationary current saturates at lower light intensities than the transient component (Fig. 2, C and D). Upon stimulation with two subsequent light pulses, the transient component of the second photocurrent is reduced in size (light adaptation; Fig. 3, A and B), and the recovery of the transient signal (dark adaptation) depends on the delay between the light pulses (Fig. 3C). The time constant ({tau}4) of the transient recovery time is about 17 s at pHo 7.5.

To further investigate the photocurrent kinetics, cells were stimulated with 10-ns laser flashes of 470-nm light (Fig. 3D). The photocurrent rose with a time constant ({tau}0) in the range of 200 µs, and the current peaked at around 1 ms after the flash and relaxed to zero biexponentially with a dominant time constant {tau}2*(>90%) of about 16 ms at pHo 7.5 and -100 mV. Repetitive laser flashes did not significantly promote light adaptation, which we considered as ideal for action spectroscopy. The normalized action spectrum recorded at pHo 7.5 is rhodopsin-shaped with a half-bandwidth of 90 nm and a maximum at 470 nm (Fig. 3E). Interestingly, upon lowering the pHo to 4, the action spectrum was shifted to 484 nm, indicating two different dark states. We further determined that VChR is permeable for H+ >> Li+ > Na+ > K+ (Fig. 3F) and poorly permeable for Ca2+ (≤10% of the Na+ conductance) and is in this respect similar to its C. reinhardtii relative ChR2.


Figure 4
View larger version (61K):
[in this window]
[in a new window]

  		FIGURE 4.
Purified VChR. SDS-PAGE (left panel) and Western blot (right panel; antibody: {alpha}-rho 1D4) of purified VChR before and after glucosidase PNGase F treatment are shown.

 


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5.
UV-visible spectroscopy of purified VChR. A, absorption spectra of purified VChR in dodecyl maltoside solution at pH 6 in the dark-adapted state (black line) and after 10 s of illumination with blue light (456 nm; red line). A 5-min incubation of VChR with 50 mM hydroxylamine in the dark led to formation of the apoprotein and retinaloxime ({lambda}max = 365 nm; dotted line). B, absorption spectra of dark-adapted VChR at different pH values, normalized at 482 nm. C, recovery of dark-adapted VChR. After illumination as in A, spectra were taken at times indicated. The calculated difference spectrum (light minus dark) was plotted on the same x axis (inset). D, recovery of dark-adapted VChR recorded at 450 nm corresponding to C.

 
Spectroscopic Characterization of VChR—To get insight into the photocyle and the gating mechanism of VChR, spectroscopic measurements were performed with purified VChR. We used COS-1 cells to express the VChR 7TM region (amino acids 1-307; see supplemental data) plus nine additional amino acids corresponding to the C terminus of bovine rhodopsin (as epitope tag (23, 28) for purification and detection by protein immunoblotting). The expressed VChR was reconstituted with all-trans-retinal and purified by immunoaffinity chromatography in dodecyl maltoside solution. Fig. 4 shows the purified protein as a double band of 31 and 34 kDa on a SDS-PAGE (left panel) and after protein immunoblotting (right panel). The 34-kDa band disappeared after treatment with glucosidase (PNGase F), suggesting that the protein was partially glycosylated.

The spectrum of dark-adapted VChR at pH 6 showed an absorption with vibrational fine structure and two maxima at 450 and 470 nm, indicating a rigid chromophore with protonated retinal-protein Schiff base linkage (Fig. 5A). The absorption is sensitive to hydroxylamine, which extracts the retinal by formation of retinaloxime (dotted line) and allows determination of the molar extinction coefficient {epsilon}470 {approx} 45.500 M·cm-1 (based on {epsilon}360 {approx} 54.000 M·cm-1 for retinal oxime) (29). Lys-252 in TM7 was identified as the site of retinal attachment, because the K252G mutant did not show any absorption beyond 400 nm (data not shown). Acidification of the medium altered the shape of the VChR spectrum asymmetrically in favor of the long wavelength peak (Fig. 5B). The dark form observed at pH 8 was named D470, whereas the acidic form was termed D480. When the sample was illuminated at pH 6 with a blue LED (456 nm) for 10 s, the absorption again shifted slightly to the red (Fig. 5, A, red versus black spectrum, and C) in a similar manner as observed after acidification. Again, the absorption slightly declined, but in this case the shift was transient with {tau} {approx} 45 s at 20 °C (Fig. 5, C and D).

Next, purified VChR was excited with 10-ns laser flashes (450 nm), and absorption changes were recorded from 300 to 600 nm in 10-nm intervals up to 150 ms after the flash. At pH 7.3, the flashes initiated a fast absorption increase between 480 and 580 nm, which reached 90% of its maximal amplitude already at the first recorded time point (50 ns after the flash). Series of absorption difference spectra at pH 7.3 for the time ranges of 0.3-15 µs and 0.033-1.5 ms after the flash are shown in Fig. 6 (A and B, respectively). The three-dimensional spectra were reconstructed using significant components identified by singular value decompositon (see "Experimental Procedures"). The fastest occurring absorption difference maximum was found at 520 nm (Fig. 6C, Early spectrum), corresponding to an absolute maximum of the photoproduct near 500 nm. Accordingly, this early photocycle intermediate was named P500. It converted with {tau} {approx} 4 µs into a species termed P390 (Fig. 6A) seen as an absorption difference peak between 370 and 380 nm in the transient spectrum (Fig. 6C, Medium spectrum). P390 decayed with {tau} {approx} 200 µs into a third photoproduct with an absorption difference maximum at 530 nm (Fig. 6, B and D, Late spectrum). This late intermediate was named P510 according to the estimated absolute absorption maximum.

At pH 4.5, flashes again initiated immediate absorption differences but with an early positive absorption difference peak at 535 nm (Fig. 6E), which is clearly red-shifted compared with the early photoproduct monitored at pH 7.3. The corresponding absorption maximum of the photoproduct is between 510 and 520 nm, and the species was termed P515 accordingly. The rise time was again below 100 ns. At later times the absorption difference maximum shifted to {approx}550 nm. This late species with a corresponding absorption maximum around 530 nm was named P530. (Fig. 6E). Unfortunately, the resolution of our detection system and the smaller absorption changes under acidic conditions did not allow observation of the transition between P515 and P530. Because no photoproduct equivalent to P390 was observed at pH 4.5, we conclude that only neutral conditions allow the formation or transient accumulation of a photoproduct with deprotonated retinal Schiff base during the VChR photocycle.


Figure 6
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 6.
Transient absorption difference spectra of purified VChR. A, three-dimensional reconstruction of typical time-resolved absorption changes at pH 7.3 in the range from 0.3 to 15 µs after laser excitation. B, reconstructed spectra at pH 7.3 for the time interval 0.033-1.5 ms after flash. C, absorption differences resulting from A. The early intermediate P500 (black circles) is seen as the difference maximum at 520 nm (arrow; Early spectrum), whereas the two later intermediates P390 and P510 are seen as difference maxima at 370 and 530 nm, respectively (blue triangles; Medium spec-trum). D, absorption differences resulting from B. Although the medium difference spectrum (blue triangles) comprises P390 and P510, the late difference spectrum shows a single photocycle intermediate P510 only (red circles). E, absorption differences after a laser flash at pH 4.5. The early intermediate P515 (black circles) is seen as the difference maximum at 535 nm (arrow), whereas the late intermediate P530 is seen as difference maximum at 550 nm (red circles; arrow). The difference spectra result from two representative data sets: 0.25-59 µs after a flash (Early spectrum) and 0.15-7.5 ms (Late spectrum).

 


Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 7.
Photocycle models. A, four-state photocycle model originally derived from photocurrent measurements on ChR1 from C. reinhardtii (21). Two dark states, D1 and D2, as well as two photoactivated conducting states, O1 and O2, were proposed. A pH-dependent equilibrium of D1 and D2 was included to explain all of the photocurrent properties of VChR. B, three-state model according to Nikolic et al. (30) including one closed dark state Dark, one conducting state O, one desensitized state Des (closed without possibility to photoconvert to O), and an additional photoreaction from Des to Dark. C, three-state model comprising a pH-dependent equilibrium between two dark states, D1 and D2, and one conducting state that is reached with different efficiency from D1 and D2. D, photocycle scheme according to model C but including the two fast spectroscopically identified photocycle intermediates and indicating the absoption maxima. The acidic forms of the intermediates are shown in green;P510 and P530 are considered as conducting states; rough time constants ({tau}-values) are given in blue.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
VChR Is Functionally Similar to Known ChRs from Chlamydomonas—We found that VChR from V. carteri is a functional relative of the two ChRs from C. reinhardtii with closer relation to ChR2. VChR shows electrical properties that are not explained by an earlier discussed three-state model with one closed dark state (Dark), one open state (O), one desensitized (closed and inactive) state (Des), and a single photochemical reaction from Dark to O (6). If the Des -> Dark reaction is slow, at high light intensities most protein would be trapped in Des, and the stationary current would decay to zero. If the Des -> Dark reaction is fast, the model fails to explain the slow recovery of the transient current. In contrast, all observed currents are fully consistent with the four-state model of Fig. 7A originally developed for ChR1 from photocurrent kinetics (21). It comprises two dark states, D1 and D2, and two conducting states, O1 and O2. On the basis of the present study, the model is extended by the pH dependence of the D1 {rightleftharpoons} D2 equilibrium of dark-adapted VChR seen as a shift of the action spectrum (Fig. 3E). According to the four-state model, both D1 and D2 convert upon light excitation into separate conducting states, O1 and O2, that are also in equilibrium (21). At neutral pH, the O2/O1 ratio is assumed to be higher than the D2/D1 ratio, resulting in an accumulation of the weakly conducting state O2 in the light and explaining the decay of the transient current to the stationary level. Nikolic et al. (30) quantitatively described the channelrhodopsin photocurrents by an extended three-state model (Fig. 7B) that was already considered earlier qualitatively (6). The "Nikolic model" assumes that the recovery of the Dark state from the (closed and desensitized) Des state is accelerated by light. The model is attractive because it describes the photocurrents adequately and involves less rate constants compared with the four-state model. The Nikolic model, however, does not explain the pH-induced shift of the action spectrum. Based on the electrical measurements and spectroscopic data generated for VChR, we are proposing here a photocycle (Fig. 7C) that is a hybrid of the four-state model (Fig. 7A) and the three-state Nikolic model (Fig. 7B). Because the acidic form and the preilluminated species (light-adapted VChR) are virtually identical, although there might be slight differences that are still unknown, we assume that D480 (D2) is an intermediate of the photocycle starting from D470 (D1). D1 and D2 are in a pH-dependent equilibrium, and both states are photoconverted into a conducting state O. The model converges with the fourstate model by assuming a fast equilibrium between O1 and O2 (representing O) and a preferential thermal reaction from O to D2 (D480). A back reaction from O to D1 (D470) is not necessarily excluded but not explicitly needed for explaining the photocurrents. The transient nature of the current is explained in the model shown in Fig. 7C by a low quantum efficiency for the D2 (D480) to O transition rather than by a second conducting state. The three perspectives (Fig. 7, A-C) are kinetically quite equivalent and describe the photocurrents equally well (results of the calculations are available on demand). The assumption of a less efficient photoconversion of D2 (D480) is supported by the small light-induced absorbance changes observed at pH 4.5. Moreover, proteorhodopsin, a microbial rhodopsin from the noncultivated marine {gamma}-protobacterium, shows a red-shifted absorption under moderately acidic conditions, and the photoisomerization is less efficient compared with that of the alkaline form (31). It is known that protonation of the retinal Schiff base counterion in microbial rhodopsins (Glu-118 in VChR) lowers the quantum efficiency of photochemical 13-trans- to cis-isomerization because of a rise of the C13-C14 rotation barrier in the excited state (32). For VChR this results in a reduced stationary photocurrent as soon as the D2/D1 equilibrium increases during illumination. The different amplitudes of the transient and stationary current even at saturating light are alternatively explained by a late photocycle intermediate because it occurs in all other microbial rhodopsins. Formation and decay of a corresponding state N would be adjusted in such a way that under all conditions, less than 60% of VChR is trapped in N.

Photocycle of VChR—The UV-visible absorption spectra presented above give a first insight into the absorption properties of the dark states and photointermediates of channelrhodopsin. The pH-dependent spectral equilibrium between D470 and D480 suggests protonation of an amino acid residue in the retinal-binding pocket. This residue is accessible from the extracellular bulk phase because the shift can be induced by extracellular acidification in oocyte experiments. At pH 7.3 the dominant photoproduct P510 is assigned as the conducting state that is relevant under most physiological conditions. The equivalent under acidic conditions is P530.

The spectroscopic observations on VChR and most electrophysiological data are summarized by an extended model as shown in Fig. 7D that is compatible with that of Fig. 7C. It comprises two dark states in equilibrium (D470 {rightleftharpoons} D480) and corresponding photoproducts. Starting from D470, the photoproduct P510 (considered as the dominant conducting state) is populated sequentially via P500 and P390. Because of the strongly blue-shifted absorption, P390 is assumed to be the only photoproduct with deprotonated retinal Schiff base. Based on the assumption that P510 is the conducting state at neutral pH, we conclude that the dark-adapted channel converts from the closed to the open state with {tau} {approx} 200 µs after light absorption (light gating). This value is compatible with the photocurrent rise, {tau}0, of the VChR currents in Xenopus oocytes (Fig. 3D) and in the living alga (2, 4). At acidic pH, a species with deprotonated retinal Schiff base (P390) is not observed, and light gating might be faster compared with alkaline pH conditions. The main intermediates P510 and P530 decay biphasically ({tau}2 {approx} 20 ms and {tau}3 {approx} 100 ms), indicating that there is a spectrally similar intermediate N.

The D480 -> D470 transition functionally reflects the light-dark adaptation process D2 -> D1 as recorded by photocurrent measurements, even if the transient photocurrent recovers three times as fast as the slow recovery of D470 after illumination with a light pulse (Fig. 5, C and D). The difference could derive from experimental conditions. The spectral data were obtained with VChR in solution, whereas photocurrents were recorded from VChR in oocyte membranes at different internal and external pH. The transition from D2 -> D1 (and thus D480 -> D470) is expected to be a two-step process as already proposed before (21) and possibly comprising an isomerization and deprotonation reaction.

The kinetic model presented in this study unifies electrophysiological data with spectral information and should help to interpret photocurrents recorded from intact algae under various physiological conditions. We expect that in algae a shift from D1 to D2 at low pHo and/or high light intensities reduces the H+ and cation influx and prevents major depolarization (3, 4).

Conclusion—The functional purification and spectroscopic characterization of VChR we report here represents a major step toward a detailed structure/function analysis of ChR. It opens the possibility of tailoring ChRs for neuroscience and medical applications. In analogy to the development of green fluorescent proteins with specific absorption/fluorescence properties, desired spectral and kinetic properties of ChR will be obtained by protein engineering and subsequent spectroscopic analysis. This will enable the application of new ChRs with distinct electrical and spectral properties to selected cell types in tissues or living animals to remotely stimulate nerves and to potentially restore color vision in mouse or even human retina (16, 20).


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grants Sfb 449 and Sfb 740 (to O. P. E.) and Sfb 498 (to P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental data. Back

2 Recipient of an Erasmus fellowship from the European Commission, Spanish Ministerio Educación y Ciencia, and Universidad Complutense Madrid. Back

1 To whom correspondence may be addressed. Tel.: 49-30-450-524-111; Fax: 49-30-450-524-952; E-mail: oliver.ernst{at}charite.de.

3 To whom correspondence may be addressed. Tel.: 49-30-2093-8681; E-mail: hegemape{at}rz.hu-berlin.de.

4 The abbreviations used are: ChR, channelrhodopsin; VChR, channelopsin from V. carteri; Chop, channelopsin; TM, transmembrane helix; dodecyl maltoside, n-dodecyl-β-d-maltoside; MOPS, 4-morpholinepropanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank B. Dick, J. Engelmann, D. Gradmann, R. Hagedorn, K. P. Hofmann, A. Koch, L. Lustres, M. Prigge, and M. Sommer for experimental support and discussion.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sineshchekov, O. A., Litvin, F. F., and Keszthelyi, L. (1990) Biophys. J. 57, 33-39
  2. Holland, E. M., Braun, F. J., Nonnengasser, C., Harz, H., and Hegemann, P. (1996) Biophys. J. 70, 924-931[Medline] [Order article via Infotrieve]
  3. Ehlenbeck, S., Gradmann, D., Braun, F. J., and Hegemann, P. (2002) Biophys. J. 82, 740-751[Medline] [Order article via Infotrieve]
  4. Braun, F. J., and Hegemann, P. (1999) Biophys. J. 76, 1668-1678[Medline] [Order article via Infotrieve]
  5. Nagel, G., Ollig, D., Fuhrmann, M., Kateriya, S., Musti, A. M., Bamberg, E., and Hegemann, P. (2002) Science 296, 2395-2398[Abstract/Free Full Text]
  6. Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P., and Bamberg, E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 13940-13945[Abstract/Free Full Text]
  7. Sineshchekov, O. A., Jung, K. H., and Spudich, J. L. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8689-8694[Abstract/Free Full Text]
  8. Hegemann, P., Gärtner, W., and Uhl, R. (1991) Biophys. J. 60, 1477-1489
  9. Lawson, M. A., Zacks, D. N., Derguini, F., Nakanishi, K., and Spudich, J. L. (1991) Biophys. J. 60, 1490-1498[Medline] [Order article via Infotrieve]
  10. Ishizuka, T., Kakuda, M., Araki, R., and Yawo, H. (2006) Neurosci. Res. 54, 85-94[CrossRef][Medline] [Order article via Infotrieve]
  11. Li, X., Gutierrez, D. V., Hanson, M. G., Han, J., Mark, M. D., Chiel, H., Hegemann, P., Landmesser, L. T., and Herlitze, S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 17816-17821[Abstract/Free Full Text]
  12. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K. (2005) Nat. Neurosci. 8, 1263-1268[CrossRef][Medline] [Order article via Infotrieve]
  13. Nagel, G., Brauner, M., Liewald, J. F., Adeishvili, N., Bamberg, E., and Gottschalk, A. (2005) Curr. Biol. 15, 2279-2284[CrossRef][Medline] [Order article via Infotrieve]
  14. Zhang, F., Wang, L. P., Brauner, M., Liewald, J. F., Kay, K., Watzke, N., Wood, P. G., Bamberg, E., Nagel, G., Gottschalk, A., and Deisseroth, K. (2007) Nature 446, 633-639[CrossRef][Medline] [Order article via Infotrieve]
  15. Schroll, C., Riemensperger, T., Bucher, D., Ehmer, J., Voller, T., Erbguth, K., Gerber, B., Hendel, T., Nagel, G., Buchner, E., and Fiala, A. (2006) Curr. Biol. 16, 1741-1747[CrossRef][Medline] [Order article via Infotrieve]
  16. Bi, A., Cui, J., Ma, Y. P., Olshevskaya, E., Pu, M., Dizhoor, A. M., and Pan, Z. H. (2006) Neuron 50, 23-33[CrossRef][Medline] [Order article via Infotrieve]
  17. Zhang, F., Wang, L. P., Boyden, E. S., and Deisseroth, K. (2006) Nat. Methods 3, 785-792[CrossRef][Medline] [Order article via Infotrieve]
  18. Herlitze, S., and Landmesser, L. T. (2007) Curr. Opin. Neurobiol. 17, 87-94[CrossRef][Medline] [Order article via Infotrieve]
  19. Hausser, M., and Smith, S. L. (2007) Nature 446, 617-619[CrossRef][Medline] [Order article via Infotrieve]
  20. Abbott, A. (2007) Nature 446, 588-589[CrossRef][Medline] [Order article via Infotrieve]
  21. Hegemann, P., Ehlenbeck, S., and Gradmann, D. (2005) Biophys. J. 89, 3911-3918[CrossRef][Medline] [Order article via Infotrieve]
  22. Tsunoda, S. P., Ewers, D., Gazzarrini, S., Moroni, A., Gradmann, D., and Hegemann, P. (2006) Biophys. J. 91, 1471-1479[CrossRef][Medline] [Order article via Infotrieve]
  23. Molday, R. S., and MacKenzie, D. (1983) Biochemistry 22, 653-660[CrossRef][Medline] [Order article via Infotrieve]
  24. Franke, R. R., Sakmar, T. P., Oprian, D. D., and Khorana, H. G. (1988) J. Biol. Chem. 263, 2119-2122[Abstract/Free Full Text]
  25. Meyer, C. K., Böhme, M., Ockenfels, A., Gärtner, W., Hofmann, K. P., and Ernst, O. P. (2000) J. Biol. Chem. 275, 19713-19718[Abstract/Free Full Text]
  26. Fritze, O., Filipek, S., Kuksa, V., Palczewski, K., Hofmann, K. P., and Ernst, O. P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2290-2295[Abstract/Free Full Text]
  27. Sineshchekov, O. A., and Spudich, J. L. (2006) in Handbook of Photosensory Receptors (Briggs, W. R., and Spudich, J. L., eds) pp. 25-42, Wiley-VCH Verlag GmbH & Co.
  28. MacKenzie, D., Arendt, A., Hargrave, P., McDowell, J. H., and Molday, R. S. (1984) Biochemistry 23, 6544-6549[CrossRef][Medline] [Order article via Infotrieve]
  29. Groenendijk, G. W., de Grip, W. J., and Daemen, F. J. (1979) Anal. Biochem. 99, 304-310[CrossRef][Medline] [Order article via Infotrieve]
  30. Nikolic, K., Degenaar, P., and Toumazou, C. (2006) in Proceedings of the 28th IEEE EMBS Annual International Conference Engineering in Medicine and Biology Society, Aug. 30-Sept. 3, 2006, New York, pp. 1626-1629
  31. Huber, R., Kohler, T., Lenz, M. O., Bamberg, E., Kalmbach, R., Engelhard, M., and Wachtveitl, J. (2005) Biochemistry 44, 1800-1806[CrossRef][Medline] [Order article via Infotrieve]
  32. Song, L., El-Sayed, M. A., and Lanyi, J. K. (1993) Science 261, 891-894[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
H. Wang, Y. Sugiyama, T. Hikima, E. Sugano, H. Tomita, T. Takahashi, T. Ishizuka, and H. Yawo
Molecular Determinants Differentiating Photocurrent Properties of Two Channelrhodopsins from Chlamydomonas
J. Biol. Chem., February 27, 2009; 284(9): 5685 - 5696.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Ritter, K. Stehfest, A. Berndt, P. Hegemann, and F. J. Bartl
Monitoring Light-induced Structural Changes of Channelrhodopsin-2 by UV-visible and Fourier Transform Infrared Spectroscopy
J. Biol. Chem., December 12, 2008; 283(50): 35033 - 35041.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
P. Berthold, S. P. Tsunoda, O. P. Ernst, W. Mages, D. Gradmann, and P. Hegemann
Channelrhodopsin-1 Initiates Phototaxis and Photophobic Responses in Chlamydomonas by Immediate Light-Induced Depolarization
PLANT CELL, June 1, 2008; 20(6): 1665 - 1677.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/3/1637    most recent
M708039200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ernst, O. P.
Right arrow Articles by Hegemann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ernst, O. P.
Right arrow Articles by Hegemann, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement