Optogenetics is a relatively new biological technique that uses light to control and monitor the behaviour of cells in living tissue, usually neurons, that have been genetically modified (using proteins) to become sensitive to light. It has revolutionized neuroscience in recent years, since researchers can now study how neurons behave in real time.

During information processing in the brain, ions flow across membranes though ion channels. In optogenetics, researchers replace these channels with light-gated channels such as the cation channels in channelrhodopsins (ChRs) 1 and 2, which can be remotely activated with light. They can thus “switch” biological signals, such as action potentials, on and off with light.

Unusual channels

ChRs are unusual channels. They belong to the large family of microbial rhodopsins and are seven-helical transmembrane proteins containing retinal as a chromophore. When they absorb light, retinal isomerization occurs, which produces intermediates that control how the channels open and close.

Since it was first extracted in 2003 from green algae, scientists have inserted ChR2 into animal cells so that they can activate and control cells, again mainly neurons, with light. They have also introduced artificially-induced mutations into the ChR2 to alter its properties – for example, to increase the amount of current it generates or to change the wavelength of light it responds to.

X-ray diffraction technique

“When illuminated, this protein allows positively charged ions to pass into the cell through the cell membrane,” explain the researchers, led by Ernst Bamberg of the Max Planck Institute of Biophysics in Frankfurt and Valentin Gordeliy of the Institut de Biologie Structurale at the University of Grenoble. “In a nerve cell, this depolarizes the membrane, mimicking the effect of a nerve impulse and causing this particular neuron to fire.”

Until now, however, researchers did not really know what the structure of ChR2 (and indeed other ChRs) looked like. Bamberg, Gordeliy and colleagues have now used X-ray diffraction to determine the detailed crystal structure of wild-type ChR2 as well as a mutant form of the protein (C128T). They used the meso-crystallization technique to obtain crystalline samples of the proteins by growing them in a “cubic lipid mesophase” – a medium that allows the protein to move freely without leaving the membrane. They then irradiated the crystals with X-rays with a wavelength of about 1 angstrom, which is a little less than the length of the bonds between the atoms in the protein.

Hydrogen bond network functions to open ChR2 channel

The team, which also includes researchers from the Moscow Institute of Physics and Technology, Forschungszentrum Jülich and the European Synchrotron Radiation Facility, observed two different dark-state conformations of ChR2 in the two protomers. While the backbone conformation was quite similar in the two samples, it found that the conformation of some amino acids and the positions of water molecules were different.

In both protomers, the researchers identified ion conduction pathways comprising four cavities separated by three gates (the extracellular gate, ECG, central gate, CG, and intracellular gate, ICG). The CG gate is further connected with two other gates through an extended hydrogen-bond network mediated by numerous water molecules. The presence of this complex network of hydrogen bonds helps one of these extra gates (the DC gate) to open the channel in ChR2.

Towards optimized tools for advanced optogenetic applications

Armed with this knowledge, the researchers, reporting their findings in Science DOI: 10.1126/science.aan8862, say that they will now be able to introduce meaningful mutations into the protein to better adjust its properties for use in specific optogenetics experiments.

Klaus Gerwert at Ruhr-Universität Bochum, who was not involved in this work, agrees: “A detailed mechanistic understanding is a prerequisite for rationally designed, optimized tools for advanced optogenetic applications, far exceeding most applications in the brain today,” he writes in a related Perspective article. “Such optogenetic tools may not only allow for the restoration of vision, but may be applied to improve other physiological processes such as hearing.”