Channelrhodopsins and Enzymerhodopsins
Algae belong to the most abundant lower
eukaryotes on this planet. They are exceptionally rich in sensory
photoreceptors which control developmental processes and orientation of the
algae in their light environment.
During the recent years we have identified
several sensory photoreceptors that are quite different from those that are
known from prokaryotes, green plants or animals.
light-activated ion channels
During the past 20 years we have studied the
visual process and the behavior of the green alga Chlamydomonas reinhardtii and its colonial relative Volvox carteri by various
biophysical techniques. Light initiates extremely fast photoreceptor currents
in the algal eye that depolarizes the cell and subsequently triggers voltagegated channels in the flagellar membrane (Fig.2). The photoreceptor is arhodopsin, the nature of which was unknown until 2002.
The extremely fast photoreceptor current P lead to the suggestion that
the photoreceptor and the channel are directly coupled and form one functional
unit without employing any chemical signaling between rhodopsin and the
channel. This principle of direct channel activation was substantiated by
identification of channelrhodopsins.
Channelrhodopsins (ChR) are
rhodopsins with intrinsic proton or cation conductance, or in other words ion
channels with intrinsic photoswitch. They define a new photoreceptor family
that has not been found in any other kingdom of life.The electrical properties
of ChRs were characterized in Xenopus oocytes after injection of thecRNA (originally started with Georg Nagel, Würzburg).The light inducedcurrents are graded with the light intensity and the membrane potential.
Theyin sign from inward to outward current after switching from positive tonegative voltage although the current of wild type ChR is inward rectifying.Upon light on the initial current partially inactivates towards a lower
So far we have characterized electrical
properties from 4 ChRs: ChR1 and ChR2 from Chlamydomonas
and VChR1 and VChR2 from Volvox carteri.
In channelrhodopsins the light switch is retinal that is covalentlylinked to the protein via a Schiff base. Light is absorbed by the retinal chromophore
and isomerizes the retinal from all-trans
into 13-cis. This isomerization initiates a cascade if conformational changes
of the protein and opening of the ion channel ca. 200 ms after a light flash. Recently, in cooperation
with the work group of Dr. Oliver Ernst VChR could be expressed in COS cells(cultivated green monkey cells) and purified by affinity chromatography. Usingtime resolved absorption spectroscopy (Fig.4) allowed the identification of
defined photoproducts that are arranged in a cyclic reaction scheme in Fig.5.
P510 is considered as the conducting state since it rises with a similar rate
as the photocurrent rises after a laser flash.
In the last three years Channelrhodopsins were
expressed in neuronal cells, tissues and living animals in order to trigger
action potentials simply by light. The new technology based on Channelrhodopsin
is named “Optogenetics”. For more information see:
Hegemann, P. (2008) Algal sensory
photoreceptors, Ann. Rev. Plant Biol. 59, 167-189.
Ernst, O. et al. (2008)
Photoactivation of Channelrhodopsin. J. Biol.
Chem. 283, 1637-1643.
Currently we are studying electrical properties
of several wild type Channelrhodopsins and many mutated Channelrhodopsin
Methods we are using:
cell culture, time resolved UV/Vis absorption
and fluorescence spectroscopy (Katja Stehfest)
two electrode voltage clamp (TEVC) (Satoshi
Tsunoda, Andre Berndt),
patch clamp and time resolved fluorescence
imaging (Matthias Prigge)
model development (Rolf Hagedorn)
PD Dr. Oliver Ernst (Charité): ChR-expression
PD Dr. Franz Bartl (Charité): FTIR-spectroscopy
Prof. Karl Deisseroth, Stanford: design of
Channelrhodopsin variants for neuroscience
If you are interested in these projects, please
or the persons indicated in brackets via our
home page under “staff”
Acetabularia Rhodopsin, a new proton pumping rhodopsin from green algae
acetabulum is a green algea typically found in subtropical see waters. Acetabularia
acetabulum is a giant single-cell organism that grows as large as ~10 cm tall, having three anatomic parts: a
bottom rhizoid resembling a set of roots, a long stalk with whorl of hairs, and
an umbrella that fuse into a cap. Its large cell volume allows performing
intercellular voltage clamp experiments that demonstrate hyperpolarization of
the plasmamenbrane by light illumination, therefore having postulated the
presence of a rhodopsin in a plant system. We cloned a cDNA that encodes a rhodopsin-related protein from this organism. The protein named
AR (Acetabularia Rhodopsin) has 279 amino acids with high homology with bacteriorhodopsin (BR).
AR was heterogously expressed in Xenopus laevis oocytes and its function
was studied by electrophysiological approach, so called
Two-Electrodes-Voltage-Clamp (TEVC). We found out that AR mediates
light-induced proton pump, in which the current is always outward directed. AR
is the first ion-pumping rhodopsin identified in a plant system. AR was induced its maximum proton conductance
by 520 nm wavelength light. The photocurrent comprises of 3 components, the initial peak
current, a, followed by steady-state level, b, and slow decay
current after light-off, c. All components are dependent on
transmembrane potential and on the intensity of applied light . Blue-light
causes a shunt of the photocycle.
1 A, Images of Acetabularia acetabulum in different stages of growth. B, AR is supposed to have 7
transmembrane helices and bind a all-trans retinal to form a chlomophore,
same as bacteriorhodopsin. It absorbs light and transport ptorons.
2 Observations of photocurrents from AR. A, Illumination of green light
mediates the current flow to outward direction in AR expressed cell (a),
while red light does not (b). No photo-induced current is seen without AR
(c). B, Photocurrent is independent from the cations and anion species, but
dependedt on pH (proton), indicating AR as a proton pump.
3 The photocurrent
comprises of 3 components, the initial peak current, a, followed by
steady-state level, b, and slow decay current after light-off, c.
All of their sizes are dependent on membrane-voltage (A) and light
intensity (B). Additional blue light reduces the photocurrent due to
accellerated photochemical conversion back to the dark state (C).
reaction scheme of AR photocycle.
Although AR is
functionally characterised in oocyte in detail, the physiological role in the
intact cell remains still open. It is enigma why light-driven proton pump
exists in an algae. Bacteriorhodopsin, one of the most studied light-driven
proton pump, creates proton gradient, which is utilized in bacteria as a main
energy source for various cellular activities such as ATP production and
flagellar movement. On the other hand, the plant has a photo synthesis system
as an energy source. Thus, it is unlikely that AR contributes for energy
producing. Further investigation is required to understand the physiological
relevance of AR.
For reference see: Tsunoda SP, Ewers D,
Gazzarrini S, Moroni A, Gradmann D, Hegemann P. (2006) H+-pumping
rhodopsin from the marine alga Acetabularia. Biophys J. 91,1471-9.
If you are interested in this project please