FOCUS_Optogenetics Custom-Tailored Molecules Chlamydomonas reinhardtii, a single-celled green alga, can’t see much at all with its eye composed solely of photosensitive rhodopsin molecules. Yet there is more to algal rhodopsin than one would expect. In recent years, it has triggered a revolution in neurobiology. Ernst Bamberg from the Max Planck Institute of Biophysics in Frankfurt helped make it famous. He is now researching these molecules and developing new variants for basic research and medical applications. n TEXT CATARINA PIETSCHMANN ll Chlamydomonas requires when light straightens out the retinal, are transduced faster into an electrical in order to see is an accu- which takes on a bent shape in the dark. signal in an algal cell than they are in mulation of proteins, known In humans and other mammals, this the human eye. as an eyespot. Under a mi- activates the rhodopsin and, via a mul- The researchers named this partic- croscope, the eyespot ap- tistage process, blocks positive ions from ular protein “channelrhodopsin.” A pears as a yellow dot in an otherwise flooding into the cell. They soon realized that this protein green algal cell. It allows Chlamydomo- harbors great potential for science. In nas to see what it needs to see – light, ALL-IN-ONE PHOTORECEPTOR a comprehensive patent specification dark, and a few shades in between – so AND ION CHANNEL published following their discovery, that the cell can swim closer to or fur- they even included a detailed list of ther away from the water surface, de- In 2002, Bamberg and Georg Nagel, to- possible applications in the fields of pending on the light conditions. gether with Peter Hegemann from neurobiology and biomedicine. “In The eyespot is composed of around Humboldt University in Berlin, discov- retrospect, that was almost a rather 200 different proteins, including pho- ered the mechanism of algal rhodop- presumptuous thing to do, but almost tosensitive rhodopsin molecules. Simi- sins. The researchers transferred the all of it has since come true. Today, lar rhodopsins can also be found in the rhodopsin gene to egg cells of a clawed there are hardly any applications for human eye, or more specifically, in the frog and observed that the proteins channelrhodopsins that aren’t includ- retinal photoreceptors, where they combine the photoreceptor and the ed in our patent,” says Ernst Bamberg. transduce the incident light into an ion channel into one single protein. To name just one example: a license electrical signal that is then transmit- The rhodopsin in algae thus has a dif- extract for treating eye diseases has al- ted to the brain for further processing. ferent function than the rhodopsins in ready been granted to a large pharma- Rhodopsins are made up of two mammals: the opsin itself forms an ceutical company. components: the protein opsin and the ion channel, which can be opened by It all sounds very simple, and with carotenoid retinal, a photosensitive mol- light so that the ions can then pass the methods of modern molecular bi- ecule. In the eye, the act of seeing starts through. As a result, the light stimuli ology, it is just that: When the gene for Graphic: Angewandte Chemie 2013-125/37/Thomas Sattig, Christian Rickert, Ernst Bamberg, Heinz Jürgen Steinhoff, Baman 26 MaxPlanckResearch 4 | 14 A channelrhodopsin-2 molecule before and after exposure to light: The protein’s amino acid chain is rolled up into a spiral measuring seven times the diameter of the cell membrane. When exposed to incident light, helix 2 (turquoise) twists out (green), opening the ion channel for calcium (green spheres) and sodium ions (turquoise spheres). In the middle of the channel, the small, photo- sensitive retinal (green/turquoise) is bound to the protein. 4 | 14 MaxPlanckResearch 27 FOCUS_Optogenetics This page: Julia Spitz and Ernst Bamberg prepare an experiment involving frog eggs. It was in this kind of cell that Bamberg first measured the electrical currents that flow through the channelrhodopsins. Opposite page: A measuring chamber for electrophysiological experiments. In such a chamber, the researchers study the channelrhodopsins in cells that are easy to handle, such as embryonic kidney cells. Using what is known as the patch clamp procedure, the scientists press a glass microelectrode (left) onto the cell surface. It records the electric current flowing through the ion channels when the cell is exposed to light from an optical fiber (right). one of the various channelrhodopsins ties years earlier. The halorhodopsin is rendered it motionless. In a parallel – channelrhodopsin-2 – is implanted light-gated as well – albeit activated by study conducted in collaboration with into a nerve cell, the cell then starts yellow light, and not by blue light like Karl Deisseroth from Stanford Univer- producing the ion channel and incor- channelrhodopsin-2. sity, the researchers showed that the porating it into its cell membrane. The Nerve cells containing the genes for two rhodopsins can also switch nerve cell can now be switched on using blue channelrhodopsin-2 and halorhodop- cells in a cell culture on and off. light, and starts producing electrical sin can therefore be switched on and stimuli. “Before, nerve cells could be off at will using light: blue light lets GREATER PHOTOSENSITIVITY, activated only with microelectrodes. positive sodium and calcium ions flow GREATER SPEED Thanks to channelrhodopsin-2, this rel- in, thus making the cell more positive, atively arduous procedure is no longer while yellow light opens the gates for Bamberg is an expert on charge trans- necessary in many neurobiological ex- negatively charged chloride ions, shift- ports via cell membranes, namely via perimental set-ups, especially in live an- ing the cell potential into the negative the barriers that serve both as a cell’s imals,” Bamberg explains. “Now, for ex- range. “One of the major advantages protective wall and as its interface with ample, it’s possible to alter the activity here is that we can simply use different the surrounding environment. One of of nerve cells in a mouse’s brain with a wavelengths of light to switch on or off the current focal points of Bamberg’s laser beam and then analyze the result- individual, electrically excitable cells, research is developing new rhodopsin ing behavior on a cellular level.” such as nerve and muscle cells in cul- variants with optimized properties. Basically, all that’s missing now is tures and in live animals, without the However, this goal required an even an off switch. As it so happens, nature need for electrodes. What’s more, we deeper understanding of how the chan- already has a solution on hand for that, can do it with much greater temporal nel works. He therefore first investigat- too: the bacterium Natronomonas phar- and spatial resolution than ever be- ed what factors determine the chan- aonis, which was discovered in an Egyp- fore,” Bamberg concludes. nel’s permeability to certain ions, and tian salt lake in the 1980s, can brave In 2005 – and also in 2007, together how the sensitivity to different wave- the high salt concentration in its habi- with Alexander Gottschalk from Frank- lengths of light influences the chan- tat only by accumulating even more furt University – Bamberg and Nagel nel’s activity. In so doing, Bamberg cre- salt inside the cell itself. Using the pho- were able to use these molecular light ated the prerequisites needed to search tosensitive ion pump halorhodopsin, it switches to control the behavior of a for rhodopsins that are permeable only actively transports negatively charged living organism with light for the first to specific ions, for example, or that are chloride ions into the cell. As long as time ever. They equipped nerve and activated by other wavelengths. the halorhodopsin is active, it remains muscle cells of the roundworm C. elegans Together with colleagues at the Max in this resting state and cannot be elec- with channelrhodopsin-2 and halo- Planck Institute in Frankfurt and from trically activated. Bamberg had already rhodopsin. Blue light induced the worm Osnabrück University, he observed studied this protein’s transport proper- to wriggle forward, while yellow light which segments of channelrhodopsin-2 Photo: Axel Griesch 28 MaxPlanckResearch 4 | 14 to-one ratio,” Ernst Bamberg explains. As a result, the activity of a cell can be switched on with blue light and switched off using yellow light under better-defined conditions and with greater precision than before. Yet Bamberg and his team don’t just develop new molecules; they are also advancing the respective range of ap- plications: Optogenetics could restore the sight of a person whose eyes no longer contain natural rhodopsin. This means, however, that the scientists would first need to conduct animal ex- periments to get other cells in the ret- ina to produce the channelrhodopsin. are needed to open the channel. “This oped this switch by fusing together one But how can genes for an algal protein gave us some clues about what channel- channelrhodopsin and one halorho- be transferred to mammals? rhodopsin needs to look like in order for dopsin molecule. An interposed pro- With viruses! More specifically, with it to acquire new properties,” Bamberg tein couples the switch proteins to- a class of viruses that has already prov- explains. The researchers achieve this gether and fixes them firmly in the cell en successful in other gene therapy ap- by specifically altering the channelrho- membrane. “When one channelrho- proaches. These viruses are loaded with dopsin gene and creating new variants dopsin and one halorhodopsin gene the channelrhodopsin gene, which they of the protein. At the same time, the sci- are introduced into the cell’s genome can then implant into a cell’s genome. entists also inspect other channelrho- separately, the cells produce different However, that doesn’t necessarily dopsins (that have since been discov- amounts of both proteins, so that one mean that the algal rhodopsin also ered in other algae species) in search of of the two is usually dominant.